Methods for treating cardiovascular disease using a soluble CTLA4 molecule

ABSTRACT

The present invention relates to compositions and methods for treating cardiovascular system diseases by administering to a subject soluble CTLA4 molecules that block endogenous B7 molecules from binding their ligands.

This invention claims the benefit of U.S. Provisional Application No.60/492,430 filed Aug. 4, 2003, whose contents are hereby incorporated byreference in their entirety.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

The present invention relates generally to the field of cardiovasculardiseases. In particular, the invention relates to methods andcompositions for treating or preventing cardiovascular diseases byadministering to a subject an effective amount of soluble CTLA4molecules alone, or in conjunction with other therapeutic agents.

BACKGROUND OF THE INVENTION

Approximately 62 million Americans have one or more types ofcardiovascular disease with coronary heart disease (CHD) and strokeafflicting more than 17 million patients in the United States alone(Ref: American Heart Association. 2002 Heart and Stroke StatisticalUpdate. Dallas, Tex.: American Heart Association; 2001). Despitenumerous therapeutic options and technological advances, morbidity andmortality from these diseases are exceedingly high; in fact,cardiovascular diseases are the leading cause of death in the UnitedStates claiming 2 of every 5 deaths. Consequently, new approaches to thetreatment and prevention of cardiovascular diseases are needed.

The concept that atherosclerosis, the leading cause of CHD, is a processof passive accumulation of lipid in arterial walls eventually leading tothe development of symptomatic cardiovascular disease is no longertenable (Ref: Scientific American 2002 (May): 46-55). Instead, thishypothesis is being replaced by evidence that cardiovascular diseaserepresents a chronic inflammatory process (Ref: Circulation 2002;105:1135-43). It has been proposed that underlying and preceding acutecoronary or cerebrovascular events are “vulnerable” (or high-risk)atherosclerotic plaque(s). Inflammation is thought to be a majorcontributing factor to plaque instability and rupture leading tounstable angina (UA) and acute myocardial infarction (AMI). Vulnerableplaques have been shown to be frequently present in numerousanatomically distinct locations rather than isolated to a single(culprit) lesion (Ref: Circulation 2003;107:2072-2075). The multifocalnature of vulnerable plaques has been documented in autopsy series andin studies using angiographic, intravascular ultrasound (IVUS),angioscopic, or thermography techniques. Clinical endpoint data (e.g.,death, myocardial infarction, stroke) that further support thesehypotheses are derived from epidemiological data, retrospectivehypothesis-generating analyses of completed clinical trials, andprospective evaluation in clinical trials (summarized below).

Epidemiological data from the Physician's Health Study providescompelling evidence that markers of inflammation (e.g., C-reactiveprotein [CRP], fibrinogen, interleukin-6, soluble intracellular adhesionmolecule-1 [sICAM-1]) are predictive of the future risk of developingAMI (Ref: Circulation 1999;100:1148-1150). Subsequently, more than adozen population-based epidemiological studies have reported similarobservations (Ref: Circulation 2002; 105:1135-43). CRP (and highsensitivity CRP [hs-CRP]) has been the most widely studied inflammatorymarker across these epidemiological studies. CRP appears to be anindependent predictor of subsequent cardiovascular events (AMI, death)in both primary prevention and secondary prevention patient populations.

Experimental and clinical evidence supports the notion that reduction ofinflammation leads to a reduction in clinical events. Aspirin has beenshown in the Physician's Health Study to be associated with a reductionof inflammation, as measured by CRP, and is associated with aconcomitant reduction of coronary events (Ref: Circulation 1999;100:1148-1150). To what extent anti-inflammatory effects/CRP reductionvs. antiplatelet effects of aspirin had on this finding in a populationof apparently healthy men is not known. Hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors (“statins”; e.g., pravastatin,simvastatin, atorvastatin, fluvastatin, and lovastatin) serve as asecond example of an existing therapy that has anti-inflammatoryproperties. In addition to their effects on serum lipids, statins alsoreduce CRP (Ref: Circulation 2002; 105:1135-43). The Pravastatin in theCholesterol and Recurrent Events (CARE) study provided the firstclinical evidence that statin therapy lowers CRP in a fashion unrelatedto low-density lipoprotein (LDL) or high-density lipoprotein (HDL)cholesterol. The magnitude of relative risk reduction for subsequentcardiovascular events in this hypercholesterolemic population wasgreater among subjects who also had evidence of inflammation (i.e., CRPelevation) as compared with those without evidence of inflammation (Ref:Circulation 1999; 100:230-235). This observation was prospectivelyvalidated in the Pravastatin Inflammation CRP Evaluation (PRINCE) studyand also reported in analysis of several other trials with a variety ofstatins (Ref: Circulation 2002; 105:1135-43).

Evidence dissociating the benefits of statin therapy in patients withelevations in CRP from those with elevation of cholesterol are derivedfrom the Air Force/Texas Coronary Atherosclerosis Prevention Study(AFCAPS/TexCAPS; Ref: New Engl J Med 2001;344:1959-1965). This study wasa primary prevention study using lovastatin in a population with low tomoderate cardiovascular risk. In this study, the magnitude of riskreduction afforded by lovastatin as compared with placebo was nearly asgreat in the patients with low levels of LDL/high levels of CRP ascompared with those with high levels of LDL/low levels of CRP.

The above findings suggest that inflammation represents a novel riskcategory that is not currently addressed as standard of care in clinicalpractice guidelines. Since approximately half of all heart attacks occurin populations with normal cholesterol levels, identification andmodulation of newly identified risk factors would be an importantstrategy for reducing cardiovascular morbidity and mortality. Thepopulation that fits within this cohort of low-LDL/high-CRP has beenestimated to be approximately 25 million Americans (Ref: Circulation2002; 105:1135-43). The elevation of CRP appears to occur in a gradedfashion consistent with subsequent risk of cardiovascular events.Specifically, elevated CRP (>3 mg/dL) is found in 10% of healthyindividuals, but is elevated in <20%, >65%, and >90% in patients withchronic stable angina, unstable angina (Braunwald class IIIb), and AMIpreceded by USA, respectively (Ref: Circulation 2002; 105:1135-43).Therefore, inflammatory markers represent an opportunity foridentification and pharmacological intervention in populations at riskfor the development of cardiovascular events.

Other markers of inflammation are emerging that may either replace oraugment the utility of CRP measurement. Recently, novel markers ofinflammation, including increases in soluble CD40 ligand (sCD40L) anddecreases in serum interleukin-10 (IL-10) concentration, have beenassociated with increased cardiovascular morbidity and mortality.Evidence suggests that CD40L is important in atherosclerotic plaquedestabilization. CD40L shed from stimulated lymphocytes ispro-inflammatory causing upregulation of inflammatory cytokines andadhesion molecules. Moreover, CD40L also promotes coagulation byinducing expression of tissue factor in macrophages and endothelialcells and also activates glycoprotein IIb/IIIa (Refs: Proc. Natl. Acad.Sci. 1997;94:1931-1936; Nature 1998;394:200-203; Circulation 2002;106:896-899).

Epidemiological evidence from the Women's Health Study demonstratedelevated serum sCD40L concentrations are associated with a graded andcontinuous rise in cardiovascular risk (Ref: Circulation2001;104:2266-2268). The increase in cardiovascular risk was nearly12-fold higher among women with the highest sCD40L concentrations.Similar findings were also observed in the c7E3 Fab Antiplatelet Therapyin Unstable Refractory Angina (CAPTURE) study (Ref: NEJM2003;348:1104-1111). In this study, a graded and continuous rise incardiovascular risk (death or nonfatal myocardial infarction) was notedamong placebo-treated subjects based upon quintiles of baseline sCD40L.This difference in cardiovascular risk was evident at both early (24hour) and late (6 month) endpoint determinations. A similar analysisfrom the CAPTURE trial investigating the anti-inflammatory cytokineIL-10 revealed that placebo-treated patients with high levels of IL-10(i.e., high anti-inflammatory cytokine levels) had a reduced risk ofdeath (Ref: Circulation 2003; 107:2109-2114). Patients in the highestquartile of serum concentrations of the anti-inflammatory cytokine IL-10had a >50% reduction in mortality rate as compared with those patientsin the lowest quartile of serum IL-10 levels. Moreover, if at the timeof hospital discharge patient populations are dichotomized as eitherhigh or low IL-10 levels there was an observed adjusted hazard ratio formortality of 0.38 (i.e., a 62% relative risk reduction) at 6 monthsfavoring subjects with high (anti-inflammatory) IL-10 levels.

In summary, the concept of cardiovascular diseases occurring as apassive process mediated by lipid deposition alone is antiquated. Thereis a substantial increase in experimental and clinical evidence thatsuggests cardiovascular disease is a manifestation of a chronicinflammatory process and that intervention in this inflammation mayreduce patient morbidity and mortality. Currently, however, the cellularand molecular mediators of these processes are only now beingelucidated. Elucidation for these processes is hampered by the lack ofan appropriate preclinical model for acute coronary syndrome (ACS).Despite the lack of knowledge of these precise mechanisms, theanti-inflammatory effects of statins appear to validate the concept thatpharmacological intervention in patients with elevated markersinflammation leads to reductions of cardiovascular morbidity andmortality. Notably, these benefits are afforded through theserendipitous pleiotropic anti-inflammatory activities of statins.Further understanding of the cellular and molecular processes may leadto the development of specific and more efficacious anti-inflammatoryagents to further reduce cardiovascular morbidity and mortality amongthese patients.

In general, the magnitude of the T-cell response is determined by theco-stimulatory response elicited by the interaction between T-cellsurface molecules and their ligands (Mueller, et al., 1989 Ann. Rev.Immunol. 7:445-480). Key co-stimulatory signals are provided by theinteraction between T-cell surface receptors, CD28 and CTLA4, and theirligands, such as B7-related molecules CD80 (i.e., B7-1) and CD86 (i.e.,B7-2), on antigen presenting cells (Linsley, P. and Ledbetter, J. 1993Ann. Rev. Immunol. 11: 191-212). T-cell activation in the absence ofco-stimulation results in anergic T-cell response (Schwartz, R. H., 1992Cell 71:1065-1068) wherein the immune system becomes nonresponsive tostimulation.

Soluble forms of CD28 and CTLA4 have been constructed by fusing variable(V)-like extracellular domains of CD28 and CTLA4 to immunoglobulin (Ig)constant domains resulting in CD28Ig and CTLA4Ig. A nucleotide and aminoacid sequence of CTLA4Ig is shown in FIG. 24 with the protein beginningwith methionine at position +1 or alanine at position −1 and ending withlysine at position +357. CTLA4Ig binds both CD80-positive andCD86-positive cells more strongly than CD28Ig (Linsley, P., et al., 1994Immunity 1:793-80). Many T-cell-dependent immune responses have beenfound to be blocked by CTLA4Ig both in vitro and in vivo. (Linsley, P.,et al., 1991b, supra; Linsley, P., et al., 1992a Science 257:792-795;Linsley, P., et al., 1992b J. Exp. Med. 176:1595-1604; Lenschow, D. J.,et al. 1992 Science 257:789-792; Tan, P., et al., 1992 J. Exp. Med.177:165-173; Turka, L. A., 1992 Proc. Natl. Acad. Sci. USA89:11102-11105).

To alter binding affinity to natural ligands, such as B7, solubleCTLA4Ig fusion molecules were modified by mutation of amino acids in theCTLA4 portion of the molecules. Regions of CTLA4 that, when mutated,alter the binding affinity or avidity for B7 ligands include thecomplementarity determining region 1 (CDR-1 as described in U.S. Pat.Nos. 6,090,914, 5,773,253, 5,844,095; in copending U.S. patentapplication Ser. No. 60/214,065; and by Peach et al, 1994. J. Exp. Med.,180:2049-2058) and complementarity determining region 3 (CDR-3)-likeregions (CDR-3 is the conserved region of the CTLA4 extracellular domainas described in U.S. Pat. Nos. 6,090,914, 5,773,253 and 5,844,095; incopending U.S. patent application Ser. No. 60/214,065; and by Peach, R.J., et al J Exp Med 1994 180:2049-2058; the CDR-3-like regionencompasses the CDR-3 region and extends, by several amino acids,upstream and/or downstream of the CDR-3 motif). The CDR-3-like regionincludes a hexapeptide motif MYPPPY (SEQ ID NO.: 20) that is highlyconserved in all CD28 and CTLA4 family members. Alanine scanningmutagenesis through the hexapeptide motif in CTLA4, and at selectedresidues in CD28Ig, reduced or abolished binding to CD80 (Peach, R. J.,et al J Exp Med 1994 180:2049-2058; U.S. Pat. No. 5,434,131; U.S. Pat.No. 6,090,914; U.S. Pat. No. 5,773,253.

Further modifications were made to soluble CTLA4Ig molecules byinterchanging homologous regions of CTLA4 and CD28. These chimericCTLA4/CD28 homologue mutant molecules identified the MYPPPY (SEQ. IDNO:20) hexapeptide motif common to CTLA4 and CD28, as well as certainnon-conserved amino acid residues in the CDR-1- and CDR-3-like regionsof CTLA4, as regions responsible for increasing the binding avidity ofCTLA4 with CD80 (Peach, R. J., et al., 1994 J Exp Med 180:2049-2058).

Soluble CTLA4 molecules, such as CTLA4Ig, CTLA4 mutant molecules orchimeric CTLA4/CD28 homologue mutants as described supra, introduce anew group of therapeutic drugs to treat cardiovascular diseases.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treatingcardiovascular diseases, by administering to a subject a molecule thatblocks B7 interactions with CTLA4 and/or CD28, thereby inhibitingendogenous B7 molecules on B7-positive cells from binding CTLA4 and/orCD28 on T-cells. Soluble CTLA4 molecules used in the methods of theinvention include CTLA4Ig and soluble CTLA4 mutant molecule L104EA29YIg.

The present invention provides compositions and methods for treatingcardiovascular diseases, by administering to a subject soluble CTLA4molecules, which bind to B7 molecules on B7-positive cells, therebyinhibiting endogenous B7 molecules from binding CTLA4 and/or CD28 onT-cells. Soluble CTLA4 molecules used in the methods of the inventioninclude CTLA4Ig and soluble CTLA4 mutant molecule L104EA29YIg.

The present invention also provides methods for treating (e.g. reducingsymptoms of) cardiovascular diseases by administering to a subjectsuffering from symptoms of cardiovascular disease, soluble CTLA4molecules such as CTLA4Ig and/or soluble CTLA4 mutant moleculeL104EA29YIg and/or a mix of any soluble CTLA molecule. CTLA4Ig and theCTLA4 mutant molecule L104EA29YIg e.g. beginning with methionine atposition +1 or alanine at position −1 and ending with lysine at position+357, as shown in FIG. 19, are preferred for use in the methods of theinvention.

The present invention also provides a pharmaceutical composition fortreating cardiovascular comprising a pharmaceutically acceptable carrierand a biologically effective agent, such as soluble CTLA4 molecules,alone or in conjunction with other therapeutic drugs.

Kits comprising pharmaceutical compositions therapeutic forcardiovascular disease are also encompassed by the invention. In oneembodiment, a kit comprising one or more of the pharmaceuticalcompositions of the invention is used to treat a cardiovascular disease.For example, the pharmaceutical composition comprises an effectiveamount of soluble CTLA4 molecules that bind to B7 molecules onB7-positive cells, thereby blocking the B7 molecules from binding CTLA4and/or CD28 on T-cells. Further, the kit may contain one or more othertherapeutic agents used in conjunction with the pharmaceuticalcompositions of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: Demographic data of patient cohorts. Demographic data includinggender, race, and disease duration as described in Example 3, infra.

FIG. 1B: Demographic data of patient cohorts. Demographic data includinggender, age, weight, and disease activity, evaluated by the patient andby the physician, as described in Example 3, infra.

FIG. 1C: Demographic data of patient cohorts as described in Example 3,infra. Demographic data including disease activity, erythrocytesedimentation rate (ESR), physical function (disability evaluated byhealth questionnaire), and C-reactive protein (CRP).

FIG. 1D: Demographic data of patient cohorts as described in Example 3,infra. Demographic data including joint swelling, joint tenderness,morning stiffness, and pain.

FIG. 1E: Demographic data of patient cohorts as described in Example 3,infra. Demographic data including prior treatments.

FIG. 2: Summary of discontinuations at day 85 by reason as described inExample 3, infra.

FIG. 3A: ACR responses at Day 85 as described in Example 3, infra:ACR-20, -50, and -70 responses.

FIG. 3B: ACR-20 responses at Day 85, including placebo response, asdescribed in Example 3, infra: ACR-20 response with 95% confidencelimits.

FIG. 3C: ACR-20 responses at Day 85 as described in Example 3, infra:Difference in ACR-20 response with respect to 95% confidence intervals.

FIG. 4A: Basic (20% improvement) clinical responses in swollen andtender joint count in percentage of patients at Day 85 as described inExample 3, infra: basic clinical response, ACR-20.

FIG. 4B: Clinical responses (in percentage improvement) in swollen andtender joint count in percentage of patients at Day 85 as described inExample 3, infra: change in clinical response in percentage improvement.

FIG. 5A: Pain response (by Likert scale by mean unit change frombaseline) in percentage of patients at Day 85 as described in Example 3,infra: pain score changes from baseline.

FIG. 5B: Patient global disease changes (by Likert scale by mean unitchange from baseline) in percentage of patients at Day 85 as describedin Example 3, infra: patient global disease activity changes.

FIG. 5C: Physician global disease changes (by Likert scale by mean unitchange from baseline) in percentage of patients at Day 85 as describedin Example 3, infra: physician global disease activity changes.

FIG. 5D: Pain (by Likert scale by mean unit change from baseline) inpercentage of patients at Day 85 as described in Example 3, infra: painchanges from baseline.

FIG. 6A: Patient global assessment of disease activity change frombaseline by range of 2 units at Day 85 as described in Example 3, infra;disease activity improvement.

FIG. 6B: Physician global assessment of disease activity change frombaseline by range of 2 units at Day 85 as described in Example 3, infra;disease activity improvement.

FIG. 7A: Percent reduction in C-reactive protein (CRP) levels at Day 85as described in Example 3, infra: percentage reduction in CRP levelsfrom baseline.

FIG. 7B: Difference in reduction in C-reactive protein (CRP) levels atDay 85 as described in Example 3, infra: percent reduction difference inCRP levels with 95% confidence intervals.

FIG. 7C: Mean reduction in C-reactive protein (CRP) levels at Day 85 asdescribed in Example 3, infra: mean change from baseline.

FIG. 8: Reduction in soluble IL-2 receptor levels mean change frombaseline at Day 85 as described in Example 3, infra.

FIG. 9A: The effect of CTLA4Ig on tender joints over time as describedin Example 3, infra: median difference from baseline.

FIG. 9B: The effect of CTLA4Ig on tender joints over time as describedin Example 3, infra: mean difference from baseline.

FIG. 10A: The effect of CTLA4Ig on swollen joints over time as describedin Example 3, infra: median difference from baseline.

FIG. 10B: The effect of CTLA4Ig on swollen joints over time as describedin Example 3, infra: mean difference from baseline.

FIG. 11: The effect of CTLA4Ig on pain assessment mean difference frombaseline over time as described in Example 3, infra.

FIG. 12A: The effect of CTLA4Ig on patient assessment of diseaseactivity mean difference from baseline over time as described in Example3, infra.

FIG. 12B: The effect of CTLA4Ig on physician assessment of diseaseactivity mean difference from baseline over time as described in Example3, infra.

FIG. 13A: The effect of L104EA29YIg on tender joints over time asdescribed in Example 3, infra: median difference from baseline.

FIG. 13B: The effect of L104EA29YIg on tender joints over time asdescribed in Example 3, infra: mean change from baseline.

FIG. 14A: The effect of L104EA29YIg on swollen joints over time asdescribed in Example 3, infra: median difference from baseline.

FIG. 14B: The effect of L104EA29YIg on swollen joints over time asdescribed in Example 3, infra: mean change from baseline.

FIG. 15: The effect of L104EA29YIg on pain assessment over time asdescribed in Example 3, infra: mean change from baseline over time.

FIG. 16A: The effect of L104EA29YIg on patient assessment of diseaseactivity mean difference from baseline over time as described in Example3, infra.

FIG. 16B: The effect of L104EA29YIg on physician assessment of diseaseactivity mean difference from baseline over time as described in Example3, infra.

FIG. 17: Percent improvement in patient disability assessed by HealthAssessment Questionnaire (HAQ) compared to the baseline at Day 85 withCTLA4Ig and L104EA29YIg treatment as described in Example 3, infra.

FIG. 18: Nucleotide and amino acid sequence of L104EIg (SEQ ID NOs: 6-7)as described in Example 1, infra.

FIG. 19: Nucleotide and amino acid sequence of L104EA29YIg (SEQ ID NOs:8-9) as described in Example 1, infra.

FIG. 20: Nucleotide and amino acid sequence of L104EA29LIg (SEQ ID NOs:10-11) as described in Example 1, infra.

FIG. 21: Nucleotide and amino acid sequence of L104EA29TIg (SEQ ID NOs:12-13) as described in Example 1, infra.

FIG. 22: Nucleotide and amino acid sequence of L104EA29WIg (SEQ ID NOs:14-15) as described in Example 1, infra.

FIG. 23: Nucleotide and amino acid sequence of CTLA4 receptor (SEQ IDNOs: 16-17).

FIG. 24: Nucleotide and amino acid sequence of CTLA4Ig (SEQ ID NOs:18-19).

FIG. 25: SDS gel (FIG. 25A) for CTLA4Ig (lane 1), L104EIg (lane 2), andL104EA29YIg (lane 3A); and size exclusion chromatographs of CTLA4Ig(FIG. 25B) and L104EA29YIg (FIG. 25C).

FIGS. 26 (left and right depictions): A ribbon diagram of the CTLA4extracellular Ig V-like fold generated from the solution structuredetermined by NMR apectroscopy. FIG. 28 (right depiction) shows anexpanded view of the CDR-1 (S25-R33) region and the MYPPPY (SEQ. IDNO:20) region indicating the location and side-chain orientation of theavidity enhancing mutations, L104 and A29.

FIGS. 27A and 27B: FACS assays showing binding of L104EA29YIg, L104EIg,and CTLA4Ig to human CD80- or CD86-transfected CHO cells as described inExample 2, infra.

FIGS. 28A and 28B: Graphs showing inhibition of proliferation ofCD80-positive and CD86-positive CHO cells as described in Example 2,infra.

FIGS. 29A and 29B: Graphs showing that L104EA29YIg is more effectivethan CTLA4Ig at inhibiting proliferation of primary and secondaryallostimulated T cells as described in Example 2, infra.

FIGS. 30A-C: Graphs illustrating that L104EA29YIg is more effective thanCTLA4Ig at inhibiting IL-2 (FIG. 30A), IL-4 (FIG. 30B), and gamma(y)-interferon (FIG. 30C) cytokine production of allostimulated human Tcells as described in Example 2, infra.

FIG. 31: A graph demonstrating that L104EA29YIg is more effective thanCTLA4Ig at inhibiting proliferation of phytohemaglutinin—(PHA)stimulated monkey T cells as described in Example 2, infra.

FIG. 32: A graph showing the equilibrium binding analysis ofL104EA29YIg, L104EIg, and wild-type CTLA4Ig to CD86Ig.

FIGS. 33A and 33B: Reduction in soluble ICAM-1 and soluble E-selectinlevels mean change from baseline at Day 85 as described in Example 3,infra.

FIG. 34: A graph showing the summary of ACR20 response by visit day inresponse to methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy, asdescribed in Example 5, infra.

FIG. 35: A graph showing the summary of ACR50 response by visit day inresponse to methotrexate alone or methotrexate and CTLA4Ig (2 and 10mg/kg) therapy, as described in Example 5, infra.

FIG. 36: A graph showing the summary of ACR70 response by visit day inresponse to methotrexate alone or methotrexate and CTLA4Ig (2 and 10mg/kg) therapy, as described in Example 5, infra.

FIG. 37: A graph showing the mean ACR-N over time in response tomethotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy,as described in Example 5, infra.

FIG. 38: A bar graph showing the ACR response in response tomethotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapyon day 180 with a 95% confidence interval, as described in Example 5,infra.

FIG. 39: A bar graph showing the proportion of New Active Joints inresponse to methotrexate alone or methotrexate and CTLA4Ig (2 and 10mg/kg) therapy on day 180, as described in Example 5, infra.

FIG. 40: A bar graph showing ACR response after therapy withmethotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) on day180, as described in Example 5, infra.

FIG. 41: A graph showing percent improvement in tender joints aftertherapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10mg/kg)—mean percent improvement from baseline, as described in Example5, infra.

FIG. 42: A graph showing percent improvement in swollen joints aftertherapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10mg/kg)—mean percent improvement from baseline, as described in Example5, infra.

FIG. 43: A graph showing percent improvement in pain after therapy withmethotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg)—meanpercent improvement from baseline, as described in Example 5, infra.

FIG. 44: A graph showing percent improvement in regard to diseaseactivity as reported by the subject after therapy with methotrexatealone or methotrexate and CTLA4Ig (2 and 10 mg/kg)—mean percentimprovement from baseline, as described in Example 5, infra.

FIG. 45: A graph showing percent improvement in regard to diseaseactivity as reported by the physician after therapy with methotrexatealone or methotrexate and CTLA4Ig (2 and 10 mg/kg)—mean percentimprovement from baseline, as described in Example 5, infra.

FIG. 46: A graph showing percent improvement regarding physical functionafter therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and10 mg/kg)—mean percent improvement from baseline as measured by HAQ, asdescribed in Example 5, infra.

FIG. 47: A graph showing percent improvement in CRP levels functionafter therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and10 mg/kg)—mean percent improvement from baseline, as described inExample 5, infra.

FIG. 48: A graph showing percent improvement in CRP levels functionafter therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and10 mg/kg)—median percent improvement from baseline, as described inExample 5, infra.

FIG. 49: A graph showing the difference in ACR response rate on day 180in two groups after therapy with CTLA4Ig (2 and 10 mg/kg) in comparisonto a group treated with methotrexate (MTX) only (95% Confidence Limits),as described in Example 5, infra.

FIG. 50: A graph showing the change from baseline for SF-36 PhysicalHealth Component on day 180, in two groups after therapy with CTLA4Ig (2and 10 mg/kg) compared to a group treated with methotrexate only (95%Confidence Limits), as described in Example 5, infra.

FIG. 51: A graph showing the change from baseline for SF-36 MentalHealth Component on Day 180, in two groups after therapy with CTLA4Ig (2and 10 mg/kg) compared to a group treated with methotrexate only (95%Confidence Limits), as described in Example 5, infra.

FIG. 52: A bar graph showing CRP levels at day 180 after therapy withmethotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), asdescribed in Example 5, infra.

FIG. 53: A bar graph showing Rheumatoid Factor levels on day 180 aftertherapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10mg/kg), as described in Example 5, infra.

FIG. 54: A bar graph showing IL-2r levels on day 180 after therapy withmethotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), asdescribed in Example 5, infra.

FIG. 55: A bar graph showing IL-6 levels on day 180 after therapy withmethotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), asdescribed in Example 5, infra.

FIG. 56: A bar graph showing TNFα levels on day 180 after therapy withmethotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), asdescribed in Example 5, infra.

FIG. 57: A table of the univariate methotrexate dose atscreening/enrollment for treatment group BMS 10—treated with CTLA4Ig at10 mg/kg body weight as described in Example 5, infra.

FIG. 58: A table of the univariate methotrexate dose atscreening/enrollment for treatment group BMS 2—treated with CTLA4Ig at 2mg/kg body weight as described in Example 5, infra.

FIG. 59: A table of the univariate methotrexate dose atscreening/enrollment for the placebo group, as described in Example 5,infra.

FIG. 60: A table of the univariate methotrexate dose up to and includingday 180 of the study for treatment group BMS 10—treated with CTLA4Ig at10 mg/kg body weight as described in Example 5, infra.

FIG. 61: A table of the univariate methotrexate dose up to and includingday 180 of the study for treatment group BMS 2—treated with CTLA4Ig at 2mg/kg body weight as described in Example 5, infra.

FIG. 62: A table of the univariate methotrexate dose up to and includingday 180 of the study for the placebo group, as described in Example 5,infra.

FIG. 63: A bar graph showing the difference in modified ACR responserates on day 180 in two groups after therapy with etanercept alone (25mg twice weekly) or in combination with CTLA4Ig (2 mg/kg), as describedin Example 6, infra.

FIG. 64A-C: Graphs showing percentage improvement of individualcomponents of the modified ACR criteria as assessed on each visit dayafter therapy with etanercept alone (25 mg twice weekly) or incombination with CTLA4Ig (2 mg/kg) as described in Example 6, infra. A.Tender Joint Count. B. Swollen Joint Count. C. Pain Assessment.

FIG. 65: A. A graph showing the change from baseline for SF-36 PhysicalHealth Component on day 180, in two groups after therapy with etanercept(25 mg biweekly) alone or in combination with CTLA4Ig (2 mg/kg) (95%Confidence Limits), as described in Example 6, infra. B. A graph showingthe change from baseline for SF-36 Mental Health Component on day 180,in two groups after therapy with etanercept (25 mg biweekly) alone or incombination with CTLA4Ig (2 mg/kg) (95% Confidence Limits), as describedin Example 6, infra.

FIG. 66: Nucleotide sequence of a CTLA4Ig encoding a signal peptide; awild type amino acid sequence of the extracellular domain of CTLA4starting at methionine at position +1 to aspartic acid at position +124,or starting at alanine at position −1 to aspartic acid at position +124;and an Ig region (SEQ ID NO.: 21).

FIG. 67: Amino acid sequence of a CTLA4Ig having a signal peptide; awild type amino acid sequence of the extracellular domain of CTLA4starting at methionine at position +1 to aspartic acid at position +124,or starting at alanine at position −1 to aspartic acid at position +124;and an Ig region (SEQ ID NO.: 22).

FIG. 68: A schematic diagram showing the disposition of subjects intothree cohorts as described in Example 7, infra.

FIG. 69: A Kaplan-Meier plot of the cumulative proportion of subjectswho discontinued for any reason during the first 12 months of the study,as described in Example 7, infra.

FIG. 70: A Kaplan-Meier plot of the cumulative proportion of subjectswho discontinued due to lack of efficacy during the first 12 months ofstudy, as described in Example 7, infra.

FIG. 71A: A graph showing the ACR Responses on Day 180 for patientsadministered methotrexate alone or methotrexate and CTLA4Ig (2 or 10mg/kg body weight) as described in Example 7, infra.

FIG. 71B: A graph showing the 95 Percent Confidence Intervals forDifferences in ACR Responses on Day 180 for patients administeredmethotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg bodyweight) as described in Example 7, infra.

FIG. 72A: A graph showing the ACR Responses on Day 360 for patientsadministered methotrexate alone or methotrexate and CTLA4Ig (2 or 10mg/kg body weight) as described in Example 7, infra.

FIG. 72B: A graph showing the 95 Percent Confidence Intervals forDifferences in ACR Responses on Day 360 for patients administeredmethotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg bodyweight) as described in Example 7, infra.

FIG. 73A: A graph summarizing the ACR 20 Response by Visit during a oneyear interval for patients administered methotrexate alone ormethotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described inExample 7, infra.

FIG. 73B: A graph summarizing the ACR 50 Response by Visit during a oneyear interval for patients administered methotrexate alone ormethotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described inExample 7, infra.

FIG. 73C: A graph summarizing the ACR 70 Response by Visit during a oneyear interval for patients administered methotrexate alone ormethotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described inExample 7, infra.

FIG. 74: A graph showing the Mean ACR-N over a one year time intervalfor patients administered methotrexate alone or methotrexate and CTLA4Ig(2 or 10 mg/kg body weight) as described in Example 7, infra.

FIG. 75: A graph showing the Proportion of New Active Joints at Day 180for patients administered methotrexate alone or methotrexate and CTLA4Ig(2 or 10 mg/kg body weight) as described in Example 7, infra.

FIG. 76A: A graph showing the Frequency of New Tender Joints per Subjectat Day 180 for patients administered methotrexate alone or methotrexateand CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7,infra.

FIG. 76B: A graph showing the Frequency of New Tender Joints per Subjectat Day 360 for patients administered methotrexate alone or methotrexateand CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7,infra.

FIG. 77A: A graph showing the Frequency of New Swollen Joints perSubject at Day 180 for patients administered methotrexate alone ormethotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described inExample 7, infra.

FIG. 77B: A graph showing the Frequency of New Swollen Joints perSubject at Day 360 for patients administered methotrexate alone ormethotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described inExample 7, infra.

FIG. 78: A graph showing the Proportion of New Active Joints at Day 360for patients administered methotrexate alone or methotrexate and CTLA4Ig(2 or 10 mg/kg body weight) as described in Example 7, infra.

FIG. 79: Graphs showing the: A) Change from Baseline in the PhysicalHealth Domains on Day 180, and B) Change from Baseline in the MentalHealth Domains on 180, for patients administered methotrexate alone ormethotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described inExample 7, infra.

FIG. 80: Graphs showing the: A) Change from Baseline in the PhysicalHealth Domains on Day 360, and B) Change from Baseline in the MentalHealth Domains on Day 360, for patients administered methotrexate aloneor methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described inExample 7, infra.

FIG. 81: A graph showing the Soluble IL-2r Levels at Baseline, Days 180and 360 for patients administered methotrexate alone or methotrexate andCTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.

FIG. 82: A graph showing the Rheumatoid Factor Levels at Baseline, Days180 and 360 for patients administered methotrexate alone or methotrexateand CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7,infra.

FIG. 83: A graph showing the ICAM-1 Levels at Baseline, Days 180 and 360for patients administered methotrexate alone or methotrexate and CTLA4Ig(2 or 10 mg/kg body weight) as described in Example 7, infra.

FIG. 84: A graph showing the e-Selectin Levels at Baseline, Days 180 and360 for patients administered methotrexate alone or methotrexate andCTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.

FIG. 85: A graph showing the Serum IL-6 at Baseline, Days 180 and 360for patients administered methotrexate alone or methotrexate and CTLA4Ig(2 or 10 mg/kg body weight) as described in Example 7, infra.

FIG. 86A: A graph showing the CRP Levels at Baseline, Days 180 and 360for patients administered methotrexate alone or methotrexate and CTLA4Ig(2 or 10 mg/kg body weight) as described in Example 7, infra.

FIG. 86B: A graph showing the TNFα Levels at Baseline, Days 180 and 360for patients administered methotrexate alone or methotrexate and CTLA4Ig(2 or 10 mg/kg body weight) as described in Example 7, infra.

FIG. 87: Percentage of activated T-cells in atherectomy specimens.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “ligand” refers to a molecule that specificallyrecognizes and binds another molecule, for example, a ligand for CTLA4is a B7 molecule. In a further example, a ligand for the B7 molecule isa CTLA4 and/or CD28 molecule. The interaction of a molecule and itsligand can be regulated by compositions of the invention. For example,CTLA4 interaction with its ligand B7 can be blocked by administration ofCTLA4Ig molecules. Alternatively, Tumor Necrosis Factor (TNF), a ligand,interacts with its receptor, the TNF receptor (TNFR), and can be blockedby administration of etanercept or other TNF/TNFR blocking molecules.

As used herein “wild type CTLA4” or “non-mutated CTLA4” has the aminoacid sequence of naturally occurring, full length CTLA4 as shown in FIG.23 (also as described in U.S. Pat. Nos. 5,434,131, 5,844,095, and5,851,795 herein incorporated by reference in their entirety), or anyportion or derivative thereof, that recognizes and binds a B7 orinterferes with a B7 so that it blocks binding to CD28 and/or CTLA4(e.g., endogenous CD28 and/or CTLA4). In particular embodiments, theextracellular domain of wild type CTLA4 begins with methionine atposition +1 and ends at aspartic acid at position +124, or theextracellular domain of wild type CTLA4 begins with alanine at position−1 and ends at aspartic acid at position +124 as shown in FIG. 23. Wildtype CTLA4 is a cell surface protein, having an N-terminal extracellulardomain, a transmembrane domain, and a C-terminal cytoplasmic domain. Theextracellular domain binds to target molecules, such as a B7 molecule.In a cell, the naturally occurring, wild type CTLA4 protein istranslated as an immature polypeptide, which includes a signal peptideat the N-terminal end. The immature polypeptide undergoespost-translational processing, which includes cleavage and removal ofthe signal peptide to generate a CTLA4 cleavage product having a newlygenerated N-terminal end that differs from the N-terminal end in theimmature form. One skilled in the art will appreciate that additionalpost-translational processing may occur, which removes one or more ofthe amino acids from the newly generated N-terminal end of the CTLA4cleavage product. Alternatively, the signal peptide may not be removedcompletely, generating molecules that begin before the common startingamino acid methionine. Thus, the mature CTLA4 protein may start atmethionine at position +1 or alanine at position −1. The mature form ofthe CTLA4 molecule includes the extracellular domain or any portionthereof, which binds to B7.

As used herein, a “CTLA4 mutant molecule” means wildtype CTLA4 as shownin FIG. 23 or any portion or derivative thereof, that has a mutation ormultiple mutations (preferably in the extracellular domain of wildtypeCTLA4). A CTLA4 mutant molecule has a sequence that it is similar butnot identical to the sequence of wild type CTLA4 molecule, but stillbinds a B7. The mutations may include one or more amino acid residuessubstituted with an amino acid having conservative (e.g., substitute aleucine with an isoleucine) or non-conservative (e.g., substitute aglycine with a tryptophan) structure or chemical properties, amino aciddeletions, additions, frameshifts, or truncations. CTLA4 mutantmolecules may include a non-CTLA4 molecule therein or attached thereto.The mutant molecules may be soluble (i.e., circulating) or bound to acell surface. Additional CTLA4 mutant molecules include those describedin U.S. patent application Ser. Nos. 09/865,321, 60/214,065 and60/287,576; in U.S. Pat. Nos. 6,090,914 5,844,095 and 5,773,253; and asdescribed by Peach, R. J., et al., in J Exp Med 180:2049-2058 (1994)).CTLA4 mutant molecules can be made synthetically or recombinantly.

“CTLA4Ig” is a soluble fusion protein comprising an extracellular domainof wildtype CTLA4 that binds B7, or a portion thereof, joined to animmunoglobulin constant region (Ig), or a portion thereof. A particularembodiment comprises the extracellular domain of wild type CTLA4 (asshown in FIG. 23) starting at methionine at position +1 and ending ataspartic acid at position +124, or starting at alanine at position −1 toaspartic acid at position +124; a junction amino acid residue glutamineat position +125; and an immunoglobulin portion encompassing glutamicacid at position +126 through lysine at position +357 (DNA encodingCTLA4Ig was deposited on May 31, 1991 with the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209under the provisions of the Budapest Treaty, and has been accorded ATCCaccession number ATCC 68629; Linsley, P., et al., 1994 Immunity1:793-80). CTLA4Ig-24, a Chinese Hamster Ovary (CHO) cell lineexpressing CTLA4Ig was deposited on May 31, 1991 with ATCCidentification number CRL-10762). The soluble CTLA4Ig molecules used inthe methods and/or kits of the invention may or may not include a signal(leader) peptide sequence. Typically, in the methods and/or kits of theinvention, the molecules do not include a signal peptide sequence.

“L104EA29YIg” is a fusion protein that is a soluble CTLA4 mutantmolecule comprising an extracellular domain of wildtype CTLA4 with aminoacid changes A29Y (a tyrosine amino acid residue substituting for analanine at position 29) and L104E (a glutamic acid amino acid residuesubstituting for a leucine at position +104), or a portion thereof thatbinds a B7 molecule, joined to an Ig tail (included in FIG. 19; DNAencoding L104EA29YIg was deposited on Jun. 20, 2000 with ATCC numberPTA-2104; copending in U.S. patent application Ser. Nos. 09/579,927,60/287,576 and 60/214,065, incorporated by reference herein). Thesoluble L104EA29YIg molecules used in the methods and/or kits of theinvention may or may not include a signal (leader) peptide sequence.Typically, in the methods and/or kits of the invention, the molecules donot include a signal peptide sequence.

As used herein, “soluble” refers to any molecule, or fragments andderivatives thereof, not bound or attached to a cell, i.e., circulating.For example, CTLA4, B7 or CD28 can be made soluble by attaching animmunoglobulin (Ig) moiety to the extracellular domain of CTLA4, B7 orCD28, respectively. Alternatively, a molecule such as CTLA4 can berendered soluble by removing its transmembrane domain. Typically, thesoluble molecules used in the methods, compositions and/or kits of theinvention do not include a signal (or leader) sequence.

As used herein, “soluble CTLA4 molecules” means non-cell-surface-bound(i.e. circulating) CTLA4 molecules or any functional portion of a CTLA4molecule that binds B7 including, but not limited to: CTLA4Ig fusionproteins (e.g. encoded by DNA deposited with ATCC accession number68629), wherein the extracellular domain of CTLA4 is fused to animmunoglobulin (Ig) moiety such as IgCγ1 (IgCgamma1), IgCγ2 (IgCgamma2),IgCγ3 (IgCgamma3), IgCγ4 (IgCgamma4), IgCμ(IgCmu), IgCα1 (IgCalpha1),IgCα2 (IgCalpha2), IgCδ(IgCdelta) or IgCεIgCepsilon), rendering thefusion molecule soluble, or fragments and derivatives thereof; proteinswith the extracellular domain of CTLA4 fused or joined with a portion ofa biologically active or chemically active protein such as thepapillomavirus E7 gene product (CTLA4-E7), melanoma-associated antigenp97 (CTLA4-p97) or HIV env protein (CTLA4-env gp120) (as described inU.S. Pat. No. 5,844,095, herein incorporated by reference in itsentirety), or fragments and derivatives thereof; hybrid (chimeric)fusion proteins such as CD28/CTLA4Ig (as described in U.S. Pat. No.5,434,131, herein incorporated by reference in its entirety), orfragments and derivatives thereof; CTLA4 molecules with thetransmembrane domain removed to render the protein soluble (Oaks, M. K.,et al., 2000 Cellular Immunology 201:144-153, herein incorporated byreference in its entirety), or fragments and derivatives thereof.“Soluble CTLA4 molecules” also include fragments, portions orderivatives thereof, and soluble CTLA4 mutant molecules, having CTLA4binding activity. The soluble CTLA4 molecules used in the methods of theinvention may or may not include a signal (leader) peptide sequence.Typically, in the methods, compositions and/or kits of the invention,the molecules do not include a signal peptide sequence.

As used herein “the extracellular domain of CTLA4” is the portion ofCTLA4 that recognizes and binds CTLA4 ligands, such as B7 molecules. Forexample, an extracellular domain of CTLA4 comprises methionine atposition +1 to aspartic acid at position +124 (FIG. 23). Alternatively,an extracellular domain of CTLA4 comprises alanine at position −1 toaspartic acid at position +124 (FIG. 23). The extracellular domainincludes fragments or derivatives of CTLA4 that bind a B7 molecule. Theextracellular domain of CTLA4 as shown in FIG. 23 may also includemutations that change the binding avidity of the CTLA4 molecule for a B7molecule.

As used herein, the term “mutation” means a change in the nucleotide oramino acid sequence of a wildtype molecule, for example, a change in theDNA and/or amino acid sequences of the wild-type CTLA4 extracellulardomain. A mutation in DNA may change a codon leading to a change in theamino acid sequence. A DNA change may include substitutions, deletions,insertions, alternative splicing, or truncations. An amino acid changemay include substitutions, deletions, insertions, additions,truncations, or processing or cleavage errors of the protein.Alternatively, mutations in a nucleotide sequence may result in a silentmutation in the amino acid sequence as is well understood in the art. Inthat regard, certain nucleotide codons encode the same amino acid.Examples include nucleotide codons CGU, CGG, CGC, and CGA encoding theamino acid, arginine (R); or codons GAU, and GAC encoding the aminoacid, aspartic acid (D). Thus, a protein can be encoded by one or morenucleic acid molecules that differ in their specific nucleotidesequence, but still encode protein molecules having identical sequences.The amino acid coding sequence is as follows:

One Letter Amino Acid Symbol Symbol Codons Alanine Ala A GCU, GCC, GCA,GCG Cysteine Cys C UGU, UGC Aspartic Acid Asp D GAU, GAC Glutamic AcidGlu E GAA, GAG Phenylalanine Phe F UUU, UUC Glycine Gly G GGU, GGC, GGA,GGG Histidine His H CAU, CAC Isoleucine Ile I AUU, AUC, AUA Lysine Lys KAAA, AAG Leucine Leu L UUA, UUG, CUU, CUC, CUA, CUG Methionine Met M AUGAsparagine Asn N AAU, AAC Proline Pro P CCU, CCC, CCA, CCG Glutamine GlnQ CAA, CAG Arginine Arg R CGU, CGC, CGA, CGG, AGA, AGG Serine Ser S UCU,UCC, UCA, UCG, AGU, AGC Threonine Thr T ACU, ACC, ACA, ACG Valine Val VGUU, GUC, GUA, GUG Tryptophan Trp W UGG Tyrosine Tyr Y UAU, UACThe mutant molecule may have one or more mutations.

As used herein, a “non-CTLA4 protein sequence” or “non-CTLA4 molecule”means any protein molecule that does not bind B7 and does not interferewith the binding of CTLA4 to its target. The non-CTLA4 molecule,attached to the extracellular domain of a CTLA4 molecule can alter thesolubility or affinity of the CTLA4 molecule. An example includes, butis not limited to, an immunoglobulin (Ig) constant region or portionthereof. Preferably, the Ig constant region is a human or monkey Igconstant region, e.g., human C(gamma)1, including the hinge, CH2 and CH3regions. The Ig constant region can be mutated to reduce its effectorfunctions (U.S. Pat. Nos. 5,637,481, 5,844,095 and 5,434,131).

As used herein, a “fragment” or “portion” is any part or segment of amolecule e.g. CTLA4 or CD28, preferably the extracellular domain ofCTLA4 or CD28 or a part or segment thereof, that recognizes and bindsits target, e.g., a B7 molecule.

As used herein, “B7” refers to the B7 family of molecules including, butnot limited to, B7-1 (CD80) (Freeman et al, 1989, J. Immunol.143:2714-2722, herein incorporated by reference in its entirety), B7-2(CD86) (Freeman et al, 1993, Science 262:909-911 herein incorporated byreference in its entirety; Azuma et al, 1993, Nature 366:76-79 hereinincorporated by reference in its entirety) that may recognize and bindCTLA4 and/or CD28. A B7 molecule can be expressed on an activated Bcell.

As used herein, “CD28” refers to the molecule that recognizes and bindsB7 as described in U.S. Pat. Nos. 5,580,756 and 5,521,288 (hereinincorporated by reference in their entirety).

As used herein, “B7-positive cells” are any cells with one or more typesof B7 molecules expressed on the cell surface.

As used herein, a “derivative” is a molecule that shares sequencesimilarity and activity of its parent molecule. For example, aderivative of CTLA4 includes a soluble CTLA4 molecule having an aminoacid sequence at least 70% similar to the extracellular domain ofwildtype CTLA4, and which recognizes and binds B7 e.g. CTLA4Ig orsoluble CTLA4 mutant molecule L104EA29YIg. A derivative means any changeto the amino acid sequence and/or chemical quality of the amino acide.g., amino acid analogs.

As used herein, to “regulate” an immune response is to activate,stimulate, up-regulate, inhibit, block, down-regulate or modify theimmune response. The cardiovascular diseases described herein, may betreated by regulating an immune response e.g., by regulating functionalCTLA4- and/or CD28-positive cell interactions with B7-positive cells.For example, a method for regulating an immune response comprisescontacting the B7-positive cells with a soluble CTLA4 molecule of theinvention so as to form soluble CTLA4/B7 complexes, the soluble CTLA4molecule interfering with reaction of an endogenous CTLA4 and/or CD28molecule with said B7 molecule.

As used herein, to “block” or “inhibit” a receptor, signal or moleculemeans to interfere with the activation of the receptor, signal ormolecule, as detected by an art-recognized test. For example, blockageof a cell-mediated immune response can be detected by determiningreduction of immune disease associated symptoms. Blockage or inhibitionmay be partial or total.

As used herein, “blocking B7 interaction” means to interfere with thebinding of B7 to its ligands, such as CD28 and/or CTLA4, therebyobstructing T-cell and B7-positive cell interactions. Examples ofmolecules that block B7 interactions with CTLA4 and/or CD28 include, butare not limited to, molecules such as an antibody (or portion orderivative thereof) that recognizes and binds to the any of CTLA4, CD28or B7 molecules (e.g. B7-1, B7-2); a soluble form (or portion orderivative thereof) of the molecules such as soluble CTLA4; a peptidefragment or other small molecule designed to interfere with the cellsignal through the CTLA4/CD28/B7-mediated interaction. In a preferredembodiment, the blocking agent is a soluble CTLA4 molecule, such asCTLA4Ig (ATCC 68629) or L104EA29YIg (ATCC PTA-2104), a soluble CD28molecule such as CD28Ig (ATCC 68628), a soluble B7 molecule such as B7Ig(ATCC 68627), an anti-B7 monoclonal antibody (e.g. ATCC HB-253, ATCCCRL-2223, ATCC CRL-2226, ATCC HB-301, ATCC HB-11341 and monoclonalantibodies as described in by Anderson et al in U.S. Pat. No. 6,113,898or Yokochi et al., 1982. J. Immun., 128(2)823-827), an anti-CTLA4monoclonal antibody (e.g. ATCC HB-304, and monoclonal antibodies asdescribed in references 82-83) and/or an anti-CD28 monoclonal antibody(e.g. ATCC HB 11944 and mAb 9.3 as described by Hansen (Hansen et al.,1980. Immunogenetics 10: 247-260) or Martin (Martin et al., 1984. J.Clin. Immun., 4(1):18-22)). Also included are small molecules that blockB7 interactions with CTLA4 and/or CD28. Blocking B7 interactions can bedetected by art-recognized tests such as determining reduction of immunedisease (e.g., cardiovascularic disease) or inflammatory diseaseassociated symptoms, by determining reduction in T-cell/B7-cellinteractions, or by determining reduction in B7 interaction with CTLA4and/or CD28. Blockage may be partial or total.

As used herein, an “effective amount” of a molecule is defined as anamount that blocks the interaction of the molecule with its ligand. Forexample, an effective amount of a molecule that blocks B7 interactionwith CTLA4 and/or CD28 may be defined as the amount of the moleculethat, when bound to B7 molecules on B7-positive cells, inhibit B7molecules from binding endogenous ligands such as CTLA4 and CD28.Alternatively, an effective amount of a molecule that blocks B7interaction with CTLA4 and/or CD28 may be defined as the amount of themolecule that, when bound to CTLA4 and/or CD28 molecules on T cells,inhibit B7 molecules from binding endogenous ligands such as CTLA4 andCD28. The inhibition or blockage may be partial or complete.

As used herein, “treating” a disease means to manage a disease bymedicinal or other therapies. Treatment of a disease may ameliorate oralleviate the symptoms of a disease, reduce the severity of a disease,alter or prevent the course of disease progression, prevent diseaseoccurrence, and/or ameliorate or alleviate or cure the basic diseaseproblem. Symptoms of cardiovascular disease include, but are not limitedto: dysrhythmias; chest pain; myocardial ischemia; angina; reducedexercise tolerance; fatigue; dyspnea on exertion; paroxysmal nocturnaldyspnea, claudication; transient ischemic attacks and quality of life.For example, to treat a cardiovascular disease may be accomplished byregulating an immune response e.g., by regulating functional CTLA4-and/or CD28-positive cell interactions with B7-positive cells.Alternatively, treating a cardiovascualar disease may be accomplished bypreventing the disease from occurring or progressing through the use ofthe compositions described herein.

As used herein, “cardiovascular disease” has the meaning commonly usedin the field, and includes, but is not limited to, the followingdiseases or conditions: thromboembolic disorders, including arterialcardiovascular thromboembolic disorders, venous cardiovascularthromboembolic disorders, and thromboembolic disorders in the chambersof the heart; ahtherosclerosis; restensosis; peripheral arterialdisease; coronary bypass grafting surgery; carotid artery disease;arteritis; myocarditis; cardiovascular inflammation; vascularinflammation; coronary heart disease (CHD); unstable angina (UA);unstable refractory angina; stable angina (SA); chronic stable angina;acute coronary syndrome (ACS); first or recurrent myocardial infarction;acute myocardial infarction (AMI); myocardial infarction; non-Q wavemyocardial infarction; non-STE myocardial infarction; coronary arterydisease; cardiac ischemia; ischemia; ischemic sudden death; transientischemic attack; stroke; atherosclerosis; peripheral occlusive arterialdisease; venous thrombosis; deep vein thrombosis; thrombophlebitis;arterial embolism; coronary arterial thrombosis; cerebral arterialthrombosis; cerebral embolism; kidney embolism; pulmonary embolism;thrombosis resulting from (a) prosthetic valves or other implants, (b)indwelling catheters, (c) stents, (d) cardiopulmonary bypass, (e)hemodialysis, or (f) other procedures in which blood is exposed to anartificial surface that promotes thrombosis; thrombosis resulting fromatherosclerosis, surgery or surgical complications, prolongedimmobilization, arterial fibrillation, congenital thrombophilia, cancer,diabetes, effects of medications or hormones, and complications ofpregnancy; cardiac arrhytmias including supraventricular arrhythmias,atrial arrhythmias, atrial flutter, atrial fibrillation; other diseaseslisted in Heart Disease: A Textbook of Cardiovascular Medicine, 2 VolumeSet, 6th Edition, 2001, Eugene Braunwald, Douglas P. Zipes, Peter Libby,Douglas D. Zipes.

Preferred cardiovascular diseases are: atherosclerosis; coronary heartdisease (CHD); restensosis; peripheral arterial disease; coronary bypassgrafting surgery; carotid artery disease; arteritis; myocarditis;cardiovascular inflammation; vascular inflammation; unstable angina(UA); unstable refractory angina; stable angina (SA); chronic stableangina; acute coronary syndrome (ACS); myocardial infarction; acutemyocardial infarction (AMI), including first or recurrent myocardialinfarction, non-Q wave myocardial infarction, non-ST elevationmyocardial infarction, and ST-segment elevation myocardial infarction.

More preferred cardiovascular diseases are: atherosclerosis; coronaryheart disease (CHD); unstable angina (UA); unstable refractory angina;stable angina (SA); chronic stable angina; acute coronary syndrome(ACS); myocardial infarction; acute myocardial infarction (AMI),including first or recurrent myocardial infarction, non-Q wavemyocardial infarction, and non-ST-segmentelevation myocardialinfarction, and ST-segment elevation myocardial infarction.

As used herein, “gene therapy” is a process to treat a disease bygenetic manipulation. Gene therapy involves introducing a nucleic acidmolecule into a cell and the cell expressing a gene product encoded bythe nucleic acid molecule. For example, as is well known by thoseskilled in the art, introducing the nucleic acid molecule into a cellmay be performed by introducing an expression vector containing thenucleic acid molecule of interest into cells ex vivo or in vitro by avariety of methods including, for example, calcium phosphateprecipitation, diethyaminoethyl dextran, polyethylene glycol (PEG),electroporation, direct injection, lipofection or viral infection(Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press 1989); Kriegler M. Gene Transfer ad Expression:A Laboratory Manual (W. H. Freeman and Co, New York, N.Y., 1993) and Wu,Methods in Enzymology (Academic Press, New York, 1993), each of which isincorporated herein by reference). Alternatively, nucleotide sequencesof interest may be introduced into a cell in vivo using a variety ofvectors and by a variety of methods including, for example: directadministration of the nucleic acid into a subject (Williams et al, 1991PNAS 88:2726-2730); or insertion of the nucleic acid molecule into aviral vector, production of the recombinant virus or viral particle, andinfection of the subject with the recombinant virus (Battleman et al,1993 J Neurosci 13:94-951; Carroll et al, 1993 J Cell Biochem 17E:241;Lebkowski et al, U.S. Pat. No. 5,354,678; Davison and Elliott, MolecularVirology: A Practical Approach (IRL Press, New York, 1993)). Othermethods used for in vivo transfer include encapsulation of the nucleicacid into liposomes, and direct introduction of the liposomes, orliposomes combined with a hemagglutinating Sendai virus, into a subject(U.S. Pat. No. 5,824,655, incorporated by reference herein). Thetransfected or infected cells express the protein products encoded bythe nucleic acid in order to ameliorate a disease or the symptoms of adisease.

As used herein, “alleviate” refers to lessening or making less severe,one or more of the symptoms of a disease, such as one or more symptomsof a cardiovascular disease, including, but not limited to:dysrhythmias; chest pain; myocardial ischemia; angina; reduced exercisetolerance; fatigue; dyspnea on exertion; paroxysmal nocturnal dyspnea,claudication; transient ischemic attacks and quality of life.

In order that the invention herein described may be more fullyunderstood the following description is set forth.

COMPOSITIONS AND METHODS OF THE INVENTION

The present invention provides compositions and methods for treatingcardiovascular diseases by administering to a subject an effectiveamount of a molecule that blocks B7 interactions with CTLA4 and/or CD28.For example, such ligands include: soluble CTLA4 molecules (such asCTLA4Ig, CTLA4-E7, CTLA4-p97, CTLA4-env gp120, and mutant CTLA4molecules such as, CTLA4/CD28Ig, L104EA29YIg, L104EA29LIg, L104EA29TIgand/or L104EA29WIg), soluble CD28 molecules, soluble B7-1 molecules,soluble B7-2 molecules, and monoclonal antibodies that recognize andbind B7, CD28 and/or CTLA4 (e.g., an anti-CTLA4 monoclonal antibody, ananti-CD28 monoclonal antibody, an anti-B7-1 monoclonal antibody or ananti-B7-2 monoclonal antibody.

An effective amount of a molecule that blocks B7 interaction with CTLA4and/or CD28 may be defined as the amount of anti-B7 monoclonalantibodies, soluble CTLA4 and/or soluble CD28 molecules that, when boundto B7 molecules on B7-positive cells, inhibit B7 molecules from bindingendogenous ligands such as CTLA4 and CD28. The inhibition may be partialor complete.

Alternatively, an effective amount of a molecule that blocks B7interaction with CTLA4 and/or CD28 may be defined as the amount ofanti-CTLA4 monoclonal antibody, anti-CD28 monoclonal antibody or solubleB7 (B7-1 or B7-2) molecules that, when bound to CTLA4 and/or CD28molecules on T cells, inhibit B7 molecules from binding endogenousligands such as CTLA4 and CD28. The inhibition may be partial orcomplete.

An effective amount of a molecule that blocks B7 interaction with CTLA4and/or CD28 is an amount about 0.1 to 100 mg/kg weight of a subject. Inanother embodiment, the effective amount is an amount about 0.5 to 100mg/kg weight of a subject, 0.5 to 5 mg/kg weight of a subject, 0.1 to 5mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject,about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight ofa subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject,about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of asubject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kgweight of a subject, about 55 to 60 mg/kg weight of a subject, about 60to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of asubject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kgweight of a subject, about 80 to 85 mg/kg weight of a subject, about 85to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of asubject, or about 95 to 100 mg/kg weight of a subject.

In an embodiment, the effective amount of a molecule that blocks B7interaction with CTLA4 and/or CD28 is an amount about 2 mg/kg to about10 mg/kg weight of a subject. The preferred amount is 10 mg/kg weight ofa subject. In another embodiment, the effective amount is an amountabout 0.1 to 4 mg/kg weight of a subject. In another embodiment theeffective amount is an amount about 0.1 to 0.5 mg/kg weight of asubject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject,about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kgweight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of asubject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject,about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kgweight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of asubject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject.

In another embodiment, the effective amount is an amount about 0.1 to 20mg/kg weight of a subject. In another embodiment, the effective amountis an amount about 0.1 to 2 mg/kg weight of a subject, about 2 to 4mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of asubject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kgweight of a subject, about 14 to 16 mg/kg weight of a subject, about 16to 18 mg/kg weight of a subject or about 18 to 20 mg/kg weight of asubject.

In another embodiment, the effective amount is about 2 mg/kg weight of asubject. In yet another embodiment, the effective amount is about 10mg/kg weight of a subject.

In a specific embodiment, the molecule that blocks B7 interaction withCTLA4 and/or CD28 is a soluble CTLA4 molecule and the effective amountof a soluble CTLA4 molecule is about 2 mg/kg weight of a subject. Inanother specific embodiment, the effective amount of a soluble CTLA4molecule is about 10 mg/kg weight of a subject. In another specificembodiment, an effective amount of a soluble CTLA4 is 500 mg for asubject weighing less than 60 kg, 750 mg for a subject weighing between60-100 kg, and 1000 mg for a subject weighing more than 100 kg.

An effective amount of the molecule that blocks B7 interaction withCTLA4 and/or CD28 is soluble CTLA4 may be administered to a subjectdaily, weekly, monthly and/or yearly, in single or multiple times perhour/day/week/month/year, depending on need. For example, in oneembodiment, the molecule may initially be administered once every twoweeks for a month, and then once every month thereafter.

In a preferred embodiment, the cardiovascular disease is:atherosclerosis; coronary heart disease (CHD); unstable angina (UA);unstable refractory angina; stable angina (SA); chronic stable angina;acute coronary syndrome (ACS); first or recurrent myocardial infarction;acute myocardial infarction (AMI); myocardial infarction; non-Q wavemyocardial infarction; or non-STE myocardial infarction.

Herein (Examples 3-7) is presented clinical data supporting the use ofmolecules that block CD28-B7 interactions in the prevention or treatmentof cardiovascular diseases. In clinical studies for RA, CTLA4Ig haslowered CRP, IL-6, and TNF-α, all markers of inflammation that alsocorrelate with cardiovascular diseases. These findings suggest that anovel mechanism of action—blocking the CD28-B7 pathway—may be useful totreat cardiovascular diseases. Further studies are discussed in Examples8 and 9. We present as our invention methods for treating cardiovasculardiseases by administering to a subject an effective amount of a moleculethat blocks B7 interactions with CTLA4 and/or CD28.

While some supporting data discussed herein for this new method oftreating cardiovascular diseases is derived from the published medicalliterature, our invention is not diminished. The concept thatimmunology/inflammation may play a critical role in cardiovasculardiseases is a relatively recent and incomplete paradigm shift. Thebreadth of research across cardiology, immunology, and infectiousdisease is substantial, so discoveries in one field do not becomeintegrated across the divide of these disciplines. Conversely, once anew discovery is made its implications into treatments and completemechanisms of actions may not be immediately transparent toinvestigators/scientists. Additionally, it has been reported in onestudy that the dominant T-cell population in patients with USA wereCD4⁺CD28^(null) cells (Ref: Circulation 2000;102:2883-2888). Thisfinding suggests that interference with CD28-B7 mediated signaling wouldnot be efficacious in UA patients.

Inhibition of CD28-B7 interaction, such as by CTLA4Ig or L104EA29YIg, toreduce cardiovascular morbidity and mortality or improve cardiovascularfunction and quality of life is new. The use of these agents across thespectrum of cardiovascular diseases is supported by: (1) cellular andbiochemical similarities between patients with rheumatoid arthritis (RA)and atherosclerosis/unstable angina; (2) the mechanism of action ofCTLA4Ig and L104EA29YIg seeming appropriate for intervention incardiovascular diseases; (3) anti-inflammatory effects of CTLA4Ig and/orL104EA29YIg in animal models and clinical studies presented herein; (4)recently reported finding on the importance of B7-CD28 interaction inatherosclerosis. Much of these data exists in disparate preclinicalstudies (cellular and animal models), as well as in clinical studiespresented herein, and is summarized below.

(1) Cellular and Biochemical Similarities Between Patients withRheumatoid Arthritis and Atherosclerosis/Unstable Angina

There are biochemical and cellular similarities between these twopathological processes (Table 1). Additionally, the premature deaths ofpatients with RA have been recently linked to acute coronary syndromes(Refs: J Rheumatology 1999;26:2562-2571; Rheumatology 1999;38:668-674).

TABLE 1 Biochemical and Cellular Similarities Between RheumatoidArthritis and Atherosclerosis/Unstable Angina Atherosclerosis/Rheumatoid Unstable Angina Arthritis Biochemical Similarities TumorNecrosis Factor-α (TNF-α) ↑ ↑ Metalloproteinase expression ↑ ↑Interleukin-6 (IL-6) ↑ ↑ C-reactive Protein (CRP) ↑ ↑↑ Adhesionmolecules (VCAM-1, ICAM- ↑ ↑ 1, E-selectin, P-selectin) Endothelin ↑ ↑Cellular Similarities Mast-cell activation ↑ ↑ T-cell activation ↑ ↑B-cell activation 0 or ↑ 0 or ↑ Possible Antigens Heat shock proteins(HSPs) Reported Reported Infections agents Reported ReportedOxidized-LDL Reported Collagen II Reported Cartilage antigens ReportedNotes: Adapted from Paceri and Teh, (Ref: Circulation 1999; 100:2124–2126). The symbols 0 denote no appreciable increase or decrease inparameter from control populations; ↑ denotes increase in parameter fromcontrol populations; Reported denotes presence of supporting evidenceavailable in the medical literature.

These similarities suggest that there may be common pathophysiologicalprocesses underlying each of these diseases.

(2) The Mechanisms of Action of CTLA4Ig and L104EA29YIg Seem Appropriatefor Intervention in Cardiovascular Diseases

T-Cells are Present in Early Atheroma as Well as Vulnerable/RupturedPlaques

There are lines of evidence that support the notion that T-cellactivation is important across the spectrum of cardiovascular diseasesfrom atherosclerosis to acute plaque rupture. T-cells are among the mostcommon cells present in human atherosclerotic lesions (Ref: New Engl JMed 1999; 340:115-126) and are present in both early lesions and invulnerable or ruptured plaques. Pathological studies of vulnerable orruptured plaques have shown that T-cells (mostly CD4⁺) are present andlocated in the shoulder region of the atheromatous lesion—the mostlikely site of tissue erosion (Refs: Am. J. Cardiol. 1991;68:36B-50B.Circulation 1994;89:36-44).

Cytokines Elaborated by T-Cells are Poised to Promote Inflammation andPlaque Rupture

T-cells are known to secrete inflammatory cytokines includinginterferon-γ (IFN-γ); TNFα, and interleukin-2 (IL-2) (Refs:Atherosclerosis 1986;6:131-138. J Clin Invest 1985;76:125-131). Theactions of T-cells in atheromatous plaques are thought to be two-fold.First, T-cells can regulate the actions of macrophages that in turn arethought to release digestive enzymes, disrupt the covering matrix andsmooth muscle layer, and promote plaque rupture. Secondly, T-cells candirectly produce a reduction of collagen synthesis in the fibrous capsof atheroma by secreting IFN-γ to vascular smooth muscle cells.Collectively, these T-cell activities are thought to represent acoordinated process that promotes atheromatous plaque instability andrupture.

Within Atheroma, T-Cells are Activated In Situ to a Degree thatParallels the Severity of Clinical Symptoms

T-cells are not only present in atheroma, but there is also evidence fortheir in situ activation within these lesions. In a study of T-cellactivation in coronary atheroma, van der Wal et. al. has shown fromatherectomy specimens that T-cells are activated as evidenced by IL-2receptor (IL-2r) expression and IL-2 secretion (Ref: Heart1998;80:14-18). The increase in T-cell expression of IL-2r in this studywas in proportion to the severity of clinical symptoms of patientsundergoing atherectomy. FIG. 87 shows the percentage of activatedT-cells in atherectomy specimens.

HSP60 or Other Novel Antigens May Directly Promote CardiovascularInflammation

Studies have suggested a possible link between infectious agents and theestablishment of cardiovascular diseases. A number of infectious agentshave been suggested including, Chlamydia pneumoniae (Cp), Helicobacterpylori, cytomegalovirus (CMV), adenoviruses, coxsackieviruses, severalHerpesviridae, and a variety of dental infections have been liked toCHD. The evidence substantiating causality among these agents withrespect to coronary atherosclerosis, however, varies widely. C.pneumoniae, a human respiratory pathogen, has most often been found inassociation with CHD.

The role of persistent elevation of CRP, and presence of antibodies toC. pneumoniae (Cp) or antibodies to human HSP60 (hu-HSP60) has beeninvestigated in a nested case-control study of the Helsinki Heart Study(Ref: Circulation 2003;107:2566-2570). This study, a prospectivedouble-blinded placebo-controlled primary prevention trial, randomized4081 subjects and observed 241 coronary events (coronary death ornon-fatal MI) over a 8.5-year follow-up. The importance of persistentseropositivity (as defined by an elevation of CRP, serum antibodies toCp or serum antibodies to hu-HSP60 at both baseline and 3 to 6 monthsprior to the index coronary event) for the prediction of subsequentcoronary events was investigated (Table 2).

TABLE 2 Odds Ratios (OR) for the Development of Coronary Death orNon-fatal MI with Persistence of Risk Factors in the Helsinki HeartStudy Unadjusted Adjusted OR (95% CI) OR (95% CI) CRP− Cp− 1 1 Cp+ 1.55(0.85-2.80) 1.62 (0.87-3.00) CRP+ Cp− 2.36 (1.16-4.79) 1.96 (0.92-4.18)Cp+ 5.38 (2.32-12.46) 4.47 (1.84-10.83) CRP− hu-HSP60− 1 1 hu-HSP60+0.92 (0.46-1.85) 0.87 (0.42-1.80) CRP+ hu-HSP60− 2.07 (1.04-4.14) 1.78(0.85-3.74) hu-HSP60+ 6.06 (2.23-16.47) 4.36 (1.53-12.39)

These data indicate that in a population of middle-aged males with noknown coronary heart disease, persistently elevated seropositivity forCp or hu-HSP60 when present with an elevated CRP, predicted subsequentcoronary events (Table 2). Of note, patients who were persistently CRP+,Cp+, and hu-HSP60+ had an adjusted OR of 16.87 (2.06-137.9) for thedevelopment of subsequent coronary events. Only 1/138 control patientsmet this criteria for a persistent elevation in all 3 markers, whereas17 were identified in index cases. These findings suggest the presenceof novel antigenic risk factors for the prospective identification ofindividuals at risk of developing cardiovascular events.

A separate recent study, reported that chlamydial heat shock protein 60(Cp-HSP60) is strongly associated with ACS (Ref: Biasucci, et. al.Circulation 2003;107:3015-3017). Unlike the asymptomatic patients in theHelsinki Heart Study, this study investigated patients at the other endof the CHD spectrum. In this study, 219 patients admitted to a CoronaryCare Unit (CCU) with either UA (Braunwald Class IIIb) or AMI werecompared to healthy patients or patients with stable angina (SA). Thepresence of an antibody response in patients to Cp-HSP60 (anti-Cp-HSP60)was measured in 100 control subjects, 40 subjects with SA, 179 subjectswith UA, and 40 subjects with AMI (Table 3).

TABLE 3 Serological Characteristics of Study Population as Reported byBiasucci, et al. Stable Unstable Control Angina Angina AMI SubjectsPatients Patients Patients (N = 100) (N = 40) (N = 179) (N = 40) Cpseropositivity 30%  60% 68% 65% HSP60 seropositivity 0% 20% 98% 99%CRP > 3 mg/dL 8% 25% 59% 62% Median CRP, mg/dL 1.4 2.1 5.05 6.02 Note:The anitbody used for HSP60 seroposivity determination in this studydoes not discriminate between Cp-HSP60 and hu-HSP60. Because of thislack of specificity these data do not support a direct associationbetween anti-HSP60 response and infection.

The above findings are supportive of the notion that a specificantigenic response exists in patients with UA and AMI. These two studiesrepresent extremes of patient populations in the spectrum ofcardiovascular disease. The Helsinki Heart Study shows that patientswithout history of coronary heart (or other major) diseases could beidentified as having an increased risk of coronary death or non-fatal MIbased upon CRP elevation and a persistent antibody response to Cp orhu-HSP60. The prevalence of these risk factors in a healthy primaryprevention population, however, was low. In stark contrast, patients inthe study by Biasucci, et. al. indicates that HSP60 seropositivity isnearly universal in patients with UA or AMI. In addition, the presenceof 20% seropositivity to HSP60 in patients with stable angina suggeststhat a graded relationship may exist between the presence of antibodiesto HSP-60 and cardiovascular events.

Antigenic Response to HSP60 is Mediated Through CD28-B7 Interactions

In animal models, macrophages (a common cell type in human atheroma)have been identified as the main inducer of IFN-γ in T-cells uponadministration of either mouse or hu-HSP60 (Ref: Int. Immunol. 2002;14:1247-1253). This process appears to occur through a classic“two-signal” model for T-cell activation involving CD28-B7 pathways. Inthis model, the activation of T-cells by either murine or hu-HSP60 isblocked by administration of CTLA4Ig (Ref: Int. Immunol. 2002;14:1247-1253). Given the high sequence homologies between human, murine,chlamydial and bacterial heat shock proteins, this finding may beconserved across species. In fact, it may be this high homology betweenthe HSPs of infectious agents and hu-HSPs that may serve to promoteautoimmunity through molecular mimicry.

HSP60 Signaling Through CD28-B7 may Afford an Opportunity forIntervention in Human Disease

There appears to be a number of similarities between preclinical modelssurrounding HSP60 and that aforementioned clinical trials (HelsinkiHeart Study; Biasucci, et. al.). First, murine models have shown thatthe strongest autoimmune response (i.e., T-cell proliferation andantibody production to self HSP60) are achieved only by concurrentadministration of both murine (self) HSP60 and Cp-HSP60 (Ref: Infectionand Immunity 1997;65:1669-1674). This preclinical finding in a unrelatedanimal model is very similar to the observed finding in the highest riskgroup in the Helsinki Heart Study. Of note, Cp-HSP60 and hu-HSP60co-localization within in human atheromatous plaques has been reported(Refs: Circulation 1998;98:300-307. J Infect Dis 1997:176:292-295). Inthese plaques, HSP60s may subsequently regulate TNF-α, matrixmetalloproteinase expression, and activate endothelium and smooth musclecells (Ref: Circulation 2003;107:3015-3017). In addition,co-administration of both murine HSP60 and Cp-HSP60 in this animal modelresults in a 6-fold decline in lymphocyte IL-10 production and a 12-foldincrease in the ratio of IFN-γ/IL-10 production. The coincident declinein IL-10 levels may translate into a clinically meaningful observationsas low serum IL-10 concentrations have been associated with aconcentration-dependent risk of death following AMI (Ref: Heeschen, et.al. Circulation 2003; 107:2109-2114). Given the parallels between theseanimal studies and data from clinical trials, inhibition of CD28-B7signaling through the use of CTLA4Ig or L104EA29YIg may represent aspecific mechanistically rational intervention for prevention and/ortreatment of cardiovascular disease.

Use of CTLA4Ig or L104EA29YIg for Prevention and/or Treatment ofCardiovascular Disease May not be Limited to HSP60

HSP60 serves as a hypothetical model for the mechanistic rationale ofCTLA4Ig or L104EA29YIg in the prevention and/or treatment ofcardiovascular disease. HSP60 however, does not represent the soleantigenic stimulation that may be interrupted by CTLA4Ig or L104EA29YIgtreatment. For example, recent epidemiological evidence from the Women'sHealth Study (Ref: Circulation 2001;104:2266-2268) and the observationaldata from the CAPTURE study (Ref: NEJM 2003;348:1104-1111) haveindependently demonstrated the existence of a graded and continuous risein cardiovascular risk (death or nonfatal myocardial infarction) basedupon serum sCD40L levels. Consequently, CD40-CD40L interactions arebecoming recognized as important mediators of inflammation incardiovascular disease. There is also evidence that suggestsinteractions between the CD28-B7 pathways and CD40-CD40L pathways inpatients with unstable angina. Specifically, it has been shown thatCD40L expressing (CD40L+) T-cells are present in atheromatous lesions(Ref: Proc. Natl. Acad. Sci. 1997;94:1931-1936). In vitro ligation ofCD40 on atheroma-associated cells has been shown to increase theproduction of pro-inflammatory cytokines, matrix metalloproteinases,adhesion molecules, and tissue factor (Ref: Nature 1998;394:200-203). Inother model systems, CD40 interaction with CD40L on antigen presentingcells (APC) increases expression of B7-1 (CD80) and B7-2 (CD86)—thestimulatory molecules for CD28-mediated T-cell activation (J. Immunol.2000;165:3506-3518). In addition, ex-vivo treatment of T-cells frompatients with unstable angina using stimulatory antibodies against CD3and CD28 stimulates the release of sCD40L (Ref: Circulation1999;100:614-620). Together, these observations may suggest that CD28-B7and CD40-CD40L signaling pathways may collectively work to promoteconditions that predispose to atherosclerotic plaque progression andrupture. Furthermore, interference with this process through use ofCTLA4Ig or L104EA29YIg may be therapeutically beneficial. Finally,other, yet undiscovered, CD28-B7-mediated processes (perhaps via novelischemia-related antigens, see below) may have important roles incardiovascular disease; use of CTLA4Ig or L104EA29YIg in these processmay be clinically useful.

(3) Anti-Inflammatory Effects of CTLA4Ig and/or L104EA29YIg in AnimalModels and Clinical Studies

Inhibition of CD28-B7 Signaling in Animal Models of Renal IschemiaSuggests that Novel Self Antigens are Important in Ischemia

The effects of CTLA4Ig on inhibition of ischemia/reperfusion injury hasbeen evaluated in a rat model (Ref: J Clin Invest 1997;100:1199-1203).In this model, rats underwent a unilateral nephrectomy and thecontralateral kidney was rendered ischemic for 45 minutes.Coadministration of CTLA4Ig, but not control immunoglobulin, largelyblocked the infiltration of CD4+ T-cells, macrophages, and majorhistocompatability (MHC) II cells into the ischemic kidney. Coincidentwith this finding, was a reduction of inflammatory “Th1” cytokines(IL-2, IFN-γ, IL2r), macrophage-associated cytokines (TNF-α. IL-6, andinducible nitric oxide synthetase), and chemoattractants/growth factors(MCP-1, RANTES, TGFβ). In addition to these findings, CTLA4Ig almostcompletely abrogates early (i.e., elevations of plasma creatinine) orlate (i.e., urine protein measurements) renal dysfunction. What isparticularly striking about this observation is the ability of CTLA4Igto inhibit this process in a model that is devoid of alloantigen.Alternatively stated, inhibition of CD28-B7 interactions in anon-transplant model of renal ischemia is afforded by CTLA4Igadministration and suggests that novel self-antigens that act throughCD28-B7 pathways are important in ischemic injury. Efficacy in thisanimal model is supportive of a role for the use of CTLA4Ig in models ofcoronary ischemia that likely has some parallel physiology.

Anti-Inflammatory Aspects of CTLA4Ig and L104EA29YIg are Consistent withTheir Use in the Prevention and/or Treatment of CV Disease

Data presented herein (Examples 3-7) for the clinical development ofCTLA4Ig and L104EA29Ig in patients with RA are encouraging with respectto favorable alterations in inflammatory markers. It is noteworthy thatpatients with RA and patients with atherosclerosis or UA have a numberof biochemical, cellular, and antigenic similarities (see above, Table1). In the phase II clinical development program in RA several markersof inflammation (CRP, IL-6, TNF-α) that are shared in patients with UAwere measured. In patients with RA, administration of CTLA4Ig at either2 mg/kg or 10 mg/kg resulted in a dose-dependent decline in CRP, IL-6,TNF-α at 180 days as compared with placebo. See FIGS. 52, 55, and 56.This effect appears to be durable and was shown to persist throughout360 days treatment with CTLA4Ig. See FIGS. 85, 86A, and 86B.

Further studies are discussed in Examples 8 and 9.

(4) Newly Reported Findings on the Importance of B7-CD28 Interactions inAtherosclerosis

Since the filing of the patent application to which this patentapplication claims priority, two reports have appeared in the scientificliterature that strongly support the role of CD28-B7 interactions in thedevelopment, progression, and instability of atheromatous lesions.First, Buono, et. al. (Circulation 2004; 109:2009-2015) characterizedthe development and progression of atheromatous lesions andplaque-antigen-specific T-cell responses in a low-density lipoproteinreceptor (LDLR)-deficient (Ldlr^(−/−)) murine model. Ldlr^(−/−) micedevelop accelerated atherosclerosis when fed a cholesterol-enricheddiet. In this study, Ldlr^(−/−) mice were crossbred withB7-1^(−/−)B7-2^(−/−) mice and the progeny were then intercrossed togenerate a compound mutant B7-1^(−/−)B7-2^(−/−) Ldlr^(−/−) line that iscompletely devoid of CD80 and CD86. This unique model allowed forinvestigation of the role of B7-CD28 interactions in an animal modelprone to develop atherosclerosis. As expected, when these animals werefed a cholesterol enriched diet, both the Ldlr^(−/−) and theB7-1^(−/−)B7-2^(−/−) Ldlr^(−/−) mice had significantly elevatedcholesterol as compared to Ldlr^(−/−) mice fed a control diet.Importantly, however, there were no differences observed between thecholesterol levels in the Ldlr^(−/−) mice fed a cholerterol enricheddiet as compared with the B7-1^(−/−)B7-2^(−/−)Ldlr^(−/−) mice fed acholesterol enriched diet. Atherosclerotic lesions in these mice werequantitated from the aortic arch and descending aorta using computerizedimage analysis from each of the 3 groups. At 8 weeks, there was minimalatherosclerosis in Ldlr^(−/−) mice receiving the control diet.Ldlr^(−/−) mice receiving a cholesterol enriched diet, however, had amarked increase in atherosclerotic lesisons in both the aortic arch anddescending aorta. In contrast, B7-1^(−/−)B7-2^(−/−) Ldlr^(−/−) micereceiving a cholesterol enriched diet had a significant reduction ofatherosclerotic lesions as compared to Ldlr^(−/−) mice receiving acholesterol enriched diet. After 20 weeks, there was progression ofatherosclerosis in all 3 treatment groups and the magnitude ofdifference observed bewteen the Ldlr^(−/−) mice and theB7-1^(−/−)B7-2^(−/−) Ldlr^(−/−) mice was somewhat diminished.Nonetheless, a statistically significant reduction in atherosclerosisbetween the Ldlr^(−/−) mice and the B7-1^(−/−)B7-2^(−/−) Ldlr^(−/−) micein the aortic arch persisted at 20 weeks.

In addition to these morphological observations, there also appears tobe functional differences between T-cells from Ldlr^(−/−) mice andB7-1^(−/−)B7-2^(−/−) Ldlr^(−/−) mice. Specifically, ex-vivo assays ofCD4+ T-cells from Ldlr^(−/−) mice and B7-1^(−/−)B7-2^(−/−) Ldlr^(−/−)mice demonstrate that only Ldlr^(−/−) mice fed a cholesterol enricheddiet produced substantial (˜5-fold higher) amounts of IFN-γ whenstimulated with mHSP60. This finding suggests that under conditions ofhypercholesterolemia and atherosclerosis, T-cells from Ldlr^(−/−) micewere primed to respond to self-HSP60.

In a second publication, Afek et. al. (Experimental and MolecularPathology 2004; 76:219-223) utilize immunohistochemical methods tocharacterize the expression of CD80 and CD86 within atheroma ofapolioprotein E knockout (ApoE^(−/−)) mice. ApoE^(−/−) mice developatherosclerosis according to their maturation age. These investigatorsevaluated the presence of CD80 and CD86 within atheromatous lesions atvarious stages of maturation. In this model, CD80 and CD86 positiveimmunostaining was present in both early and more advanced atheromatouslesions. Moreover, CD80 and CD86 staining was found to be colocalizedwith oxidized LDL—a putative autoantigen for T-cell activation.

Collectively, these two literature reports support and extend thematerial previously presented in the priority patent application. First,the notion that T-cells are present in atheroma and are activiated insitu is strengthed by the finding of CD80 and CD86 colocalization withinboth early and advanced atheroma. Second, the data obtained from theB7-1^(−/−)B7-2^(−/−) Ldlr^(−/−) mice clearly and elegantly demonstratethat interruption of B7-CD28 signaling influences the development andprogression of atherosclerotic disease in a well accepted model ofatherosclerosis. Finally, the observation that ex-vivo stimulation ofCD4+ T-cells from mHSP60 results in substantial IFN-γ productionsupports the prior hypothesis that HSP60 serve as a novel antigen topromote cardiovascular inflammation and plaque instability (i.e.,through the effects of IFN-γ on matrix metalloproteinases and collagensynthesis) through B7-CD28-dependent processes.

Compositions

The present invention provides compositions for treating cardiovasculardiseases comprising molecules that block B7 interactions with CTLA4and/or CD28, such as soluble CTLA4 molecules. Examples of soluble CTLA4include CTLA4Ig (FIG. 24) and soluble CTLA4 mutant molecules such asL104EA29YIg (FIG. 19), L104EA29LIg (FIG. 20), L104EA29Tig (FIG. 21), andL104EA29WIg (FIG. 22).

CTLA4 molecules, with mutant or wildtype sequences, may be renderedsoluble by deleting the CTLA4 transmembrane segment (Oaks, M. K., etal., 2000 Cellular Immunology 201:144-153).

Alternatively, soluble CTLA4 molecules, with mutant or wildtypesequences, may be fusion proteins, wherein the CTLA4 molecules are fusedto non-CTLA4 moieties such as immunoglobulin (Ig) molecules that renderthe CTLA4 molecules soluble. For example, a CTLA4 fusion protein mayinclude the extracellular domain of CTLA4 fused to an immunoglobulinconstant domain, resulting in a CTLA4Ig molecule (FIG. 24) (Linsley, P.S., et al., 1994 Immunity 1:793-80). Examples of immunoglobulin domainsthat may be fused to CTLA4 include, but are not limited to IgCγ1(IgCgamma1), IgCγ2 (IgCgamma2), IgCγ3 (IgCgamma3), IgCγ4 (IgCgamma4),IgCμ (IgCmu), IgCα1 (IgCalpha1), IgCα2 (IgCalpha2), IgCδ (IgCdelta) orIgCε (IgCepsilon).

For clinical protocols, it is preferred that the immunoglobulin moietydoes not elicit a detrimental immune response in a subject. Thepreferred moiety is the immunoglobulin constant region, including thehuman or monkey immunoglobulin constant regions. One example of asuitable immunoglobulin region is human Cγ1, including the hinge, CH2and CH3 regions which can mediate effector functions such as binding toFc receptors, mediating complement-dependent cytotoxicity (CDC), ormediate antibody-dependent cell-mediated cytotoxicity (ADCC). Theimmunoglobulin moiety may have one or more mutations therein, (e.g., inthe CH2 domain, to reduce effector functions such as CDC or ADCC) wherethe mutation modulates the binding capability of the immunoglobulin toits ligand, by increasing or decreasing the binding capability of theimmunoglobulin to Fc receptors. For example, mutations in theimmunoglobulin moiety may include changes in any or all its cysteineresidues within the hinge domain, for example, the cysteines atpositions +130, +136, and +139 are substituted with serine (FIG. 24).The immunoglobulin moiety may also include the proline at position +148substituted with a serine, as shown in FIG. 24. Further, the mutationsin the immunoglobulin moiety may include having the leucine at position+144 substituted with phenylalanine, leucine at position +145substituted with glutamic acid, or glycine at position +147 substitutedwith alanine.

Additional non-CTLA4 moieties for use in the soluble CTLA4 molecules orsoluble CTLA4 mutant molecules include, but are not limited to, p97molecule, env gp120 molecule, E7 molecule, and ova molecule (Dash, B. etal. 1994 J. Gen. Virol. 75 (Pt 6):1389-97; Ikeda, T., et al. 1994 Gene138(1-2):193-6; Falk, K., et al. 1993 Cell. Immunol. 150(2):447-52;Fujisaka, K. et al. 1994 Virology 204(2):789-93). Other molecules arealso possible (Gerard, C. et al. 1994 Neuroscience 62(3):721; Byrn, R.et al. 1989 63(10):4370; Smith, D. et al. 1987 Science 238:1704; Lasky,L. 1996 Science 233:209).

The soluble CTLA4 molecule of the invention can include a signal peptidesequence linked to the N-terminal end of the extracellular domain of theCTLA4 portion of the molecule. The signal peptide can be any sequencethat will permit secretion of the molecule, including the signal peptidefrom oncostatin M (Malik, et al., (1989) Molec. Cell. Biol. 9:2847-2853), or CD5 (Jones, N. H. et al., (1986) Nature 323:346-349), orthe signal peptide from any extracellular protein. The soluble CTLA4molecule of the invention can include the oncostatin M signal peptidelinked at the N-terminal end of the extracellular domain of CTLA4, andthe human immunoglobulin molecule (e.g., hinge, CH2 and CH3) linked tothe C-terminal end of the extracellular domain (wildtype or mutated) ofCTLA4. This molecule includes the oncostatin M signal peptideencompassing an amino acid sequence having methionine at position −26through alanine at position −1, the CTLA4 portion encompassing an aminoacid sequence having methionine at position +1 through aspartic acid atposition +124, a junction amino acid residue glutamine at position +125,and the immunoglobulin portion encompassing an amino acid sequencehaving glutamic acid at position +126 through lysine at position +357.

Specifically, the soluble CTLA4 mutant molecules of the invention,comprising the mutated CTLA4 sequences described infra, can be fusionmolecules comprising human Ig, e.g., IgC(gamma)1 (i.e. IgCγ1) moietiesfused to the mutated CTLA4 fragments.

In one embodiment, the soluble CTLA4 mutant molecules comprise IgCγ1(IgCgamma1) fused to an extracellular domain of CTLA4 comprising asingle-site mutation in the extracellular domain. The extracellulardomain of CTLA4 comprises methionine at position +1 through asparticacid at position +124 (e.g., FIG. 23). The extracellular domain of theCTLA4 can comprise alanine at position −1 through aspartic acid atposition +124 (e.g., FIG. 23). Examples of single-site mutations includethe following wherein the leucine at position +104 is changed to anyother amino acid:

Single-site mutant Codon change L104EIg Glutamic acid GAG L104SIg SerineAGT L104TIg Threonine ACG L104AIg Alanine GCG L104WIg Tryptophan TGGL104QIg Glutamine CAG L104KIg Lysine AAG L104RIg Arginine CGG L104GIgGlycine GGG

Further, the invention provides mutant molecules having theextracellular domain of CTLA4 with two mutations, fused to an Ig Cγ1(IgCgamma1) moiety. Examples include the following wherein the leucineat position +104 is changed to another amino acid (e.g. glutamic acid)and the glycine at position +105, the serine at position +25, thethreonine at position +30 or the alanine at position +29 is changed toany other amino acid:

Double-site mutants Codon change L104EG105FIg Phenylalanine TTCL104EG105WIg Tryptophan TGG L104EG105LIg Leucine CTT L104ES25RIgArginine CGG L104ET30GIg Glycine GGG L104ET30NIg Asparagine AATL104EA29YIg Tyrosine TAT L104EA29LIg Leucine TTG L104EA29TIg ThreonineACT L104EA29WIg Tryptophan TGG

Further still, the invention provides mutant molecules having theextracellular domain of CTLA4 comprising three mutations, fused to anIgCγ1 (IgCgamma1) moiety. Examples include the following wherein theleucine at position +104 is changed to another amino acid (e.g. glutamicacid), the alanine at position +29 is changed to another amino acid(e.g. tyrosine) and the serine at position +25 is changed to anotheramino acid:

Triple-site Mutants Codon changes L104EA29YS25KIg Lysine AAAL104EA29YS25KIg Lysine AAG L104EA29YS25NIg Asparagine AACL104EA29YS25RIg Arginine CGG

Soluble CTLA4 mutant molecules may have a junction amino acid residuewhich is located between the CTLA4 portion and the Ig portion of themolecule. The junction amino acid can be any amino acid, includingglutamine. The junction amino acid can be introduced by molecular orchemical synthesis methods known in the art.

The soluble CTLA4 proteins of the invention, and fragments thereof, canbe generated by chemical synthesis methods. The principles of solidphase chemical synthesis of polypeptides are well known in the art andmay be found in general texts relating to this area (Dugas, H. andPenney, C. 1981 Bioorganic Chemistry, pp 54-92, Springer-Verlag, NewYork). The soluble CTLA4 proteins may be synthesized by solid-phasemethodology utilizing an Applied Biosystems 430A peptide synthesizer(Applied Biosystems, Foster City, Calif.) and synthesis cycles suppliedby Applied Biosystems. Protected amino acids, such ast-butoxycarbonyl-protected amino acids, and other reagents arecommercially available from many chemical supply houses.

The present invention provides CTLA4 mutant molecules including a signalpeptide sequence linked to the N-terminal end of the extracellulardomain of the CTLA4 portion of the mutant molecule. The signal peptidecan be any sequence that will permit secretion of the mutant molecule,including the signal peptide from oncostatin M (Malik, et al., 1989Molec. Cell. Biol. 9: 2847-2853), or CD5 (Jones, N. H. et al., 1986Nature 323:346-349), or the signal peptide from any extracellularprotein.

The invention provides soluble CTLA4 mutant molecules comprising asingle-site mutation in the extracellular domain of CTLA4 such asL104EIg (as included in FIG. 18) or L104SIg, wherein L104EIg and L104SIgare mutated in their CTLA4 sequences so that leucine at position +104 issubstituted with glutamic acid or serine, respectively. The single-sitemutant molecules further include CTLA4 portions encompassing methionineat position +1 through aspartic acid at position +124, a junction aminoacid residue glutamine at position +125, and an immunoglobulin portionencompassing glutamic acid at position +126 through lysine at position+357. The immunoglobulin portion of the mutant molecule may also bemutated so that the cysteines at positions +130, +136, and +139 aresubstituted with serine, and the proline at position +148 is substitutedwith serine. Alternatively, the single-site soluble CTLA4 mutantmolecule may have a CTLA4 portion encompassing alanine at position −1through aspartic acid at position +124.

The invention provides soluble CTLA4 mutant molecules comprising adouble-site mutation in the extracellular domain of CTLA4, such asL104EA29YIg, L104EA29LIg, L104EA29TIg or L104EA29WIg, wherein leucine atposition +104 is substituted with a glutamic acid and alanine atposition +29 is changed to tyrosine, leucine, threonine and tryptophan,respectively. The sequences for L104EA29YIg, L104EA29LIg, L104EA29TIgand L104EA29WIg, starting at methionine at position +1 and ending withlysine at position +357, plus a signal (leader) peptide sequence areshown in FIGS. 19-22 respectively. The double-site mutant moleculesfurther comprise CTLA4 portions encompassing methionine at position +1through aspartic acid at position +124, a junction amino acid residueglutamine at position +125, and an immunoglobulin portion encompassingglutamic acid at position +126 through lysine at position +357. Theimmunoglobulin portion of the mutant molecule may also be mutated, sothat the cysteines at positions +130, +136, and +139 are substitutedwith serine, and the proline at position +148 is substituted withserine. Alternatively, these mutant molecules can have a CTLA4 portionencompassing alanine at position −1 through aspartic acid at position+124.

The invention provides soluble CTLA4 mutant molecules comprising adouble-site mutation in the extracellular domain of CTLA4, such asL104EG105FIg, L104EG105WIg and L104EG105LIg, wherein leucine at position+104 is substituted with a glutamic acid and glycine at position +105 issubstituted with phenylalanine, tryptophan and leucine, respectively.The double-site mutant molecules further comprise CTLA4 portionsencompassing methionine at position +1 through aspartic acid at position+124, a junction amino acid residue glutamine at position +125, and animmunoglobulin portion encompassing glutamic acid at position +126through lysine at position +357. The immunoglobulin portion of the mayalso be mutated, so that the cysteines at positions +130, +136, and +139are substituted with serine, and the proline at position +148 issubstituted with serine. Alternatively, these mutant molecules can havea CTLA4 portion encompassing alanine at position −1 through asparticacid at position +124.

The invention provides L104ES25RIg which is a double-site mutantmolecule comprising a CTLA4 portion encompassing methionine at position+1 through aspartic acid at position +124, a junction amino acid residueglutamine at position +125, and the immunoglobulin portion encompassingglutamic acid at position +126 through lysine at position +357. Theportion having the extracellular domain of CTLA4 is mutated so thatserine at position +25 is substituted with arginine, and leucine atposition +104 is substituted with glutamic acid. Alternatively,L104ES25RIg can have a CTLA4 portion encompassing alanine at position −1through aspartic acid at position +124.

The invention provides soluble CTLA4 mutant molecules comprising adouble-site mutation in the extracellular domain of CTLA4, such asL104ET30GIg and L104ET30NIg, wherein leucine at position +104 issubstituted with a glutamic acid and threonine at position +30 issubstituted with glycine and asparagine, respectively. The double-sitemutant molecules further comprise CTLA4 portions encompassing methionineat position +1 through aspartic acid at position +124, a junction aminoacid residue glutamine at position +125, and an immunoglobulin portionencompassing glutamic acid at position +126 through lysine at position+357. The immunoglobulin portion of the mutant molecule may also bemutated, so that the cysteines at positions +130, +136, and +139 aresubstituted with serine, and the proline at position +148 is substitutedwith serine. Alternatively, these mutant molecules can have a CTLA4portion encompassing alanine at position −1 through aspartic acid atposition +124.

The invention provides soluble CTLA4 mutant molecules comprising atriple-site mutation in the extracellular domain of CTLA4, such asL104EA29YS25KIg, L104EA29YS25NIg, L104EA29YS25RIg, wherein leucine atposition +104 is substituted with a glutamic acid, alanine at position+29 is substituted with tyrosine, and serine at position +25 issubstituted with lysine, asparagine and arginine, respectively. Thetriple-site mutant molecules further comprise CTLA4 portionsencompassing methionine at position +1 through aspartic acid at position+124, a junction amino acid residue glutamine at position +125, and animmunoglobulin portion encompassing glutamic acid at position +126through lysine at position +357. The immunoglobulin portion of themutant molecule may also be mutated, so that the cysteines at positions+130, +136, and +139 are substituted with serine, and the proline atposition +148 is substituted with serine. Alternatively, these mutantmolecules can have a CTLA4 portion encompassing alanine at position −1through aspartic acid at position +124.

Additional embodiments of soluble CTLA4 mutant molecules includechimeric CTLA4/CD28 homologue mutant molecules that bind a B7 (Peach, R.J., et al., 1994 J Exp Med 180:2049-2058). Examples of these chimericCTLA4/CD28 mutant molecules include HS1, HS2, HS3, HS4, HS5, HS6, HS4A,HS4B, HS7, HS8, HS9, HS10, HS11, HS12, HS13 and HS14 (U.S. Pat. No.5,773,253).

Preferred embodiments of the invention are soluble CTLA4 molecules suchas CTLA4Ig (as shown in FIG. 24, starting at methionine at position +1and ending at lysine at position +357) and soluble CTLA4 mutantL104EA29YIg (as shown in FIG. 19, starting at methionine at position +1and ending at lysine at position +357).

The invention further provides nucleic acid molecules comprisingnucleotide sequences encoding the amino acid sequences corresponding tothe soluble CTLA4 molecules of the invention. In one embodiment, thenucleic acid molecule is a DNA (e.g., cDNA) or a hybrid thereof. Forexample, a CTLA4Ig molecule can comprise a GCT or GCC codon, encodingalanine, at nucleotide position +49 to +51 as shown in FIG. 24. Inanother example, a CTLA4Ig molecule can comprise a GGT or GGG codon,encoding glycine, at nucleotide position +436 to +438 as shown in FIG.24. In yet another example, a CTLA4Ig molecule can comprise a CGG or CGTcodon, encoding arginine, at nucleotide position +631 to +633 as shownin FIG. 24. DNA encoding CTLA4Ig (FIG. 24) was deposited on May 31, 1991with the American Type Culture Collection (ATCC), 10801 UniversityBlvd., Manassas, Va. 20110-2209 and has been accorded ATCC accessionnumber ATCC 68629. DNA encoding L104EA29YIg (sequence included in FIG.19) was deposited on Jun. 19, 2000 with ATCC and has been accorded ATCCaccession number PTA-2104. Alternatively, the nucleic acid molecules areRNA or a hybrid thereof.

The nucleic acid molecules of the invention also include derivativenucleic acid molecules which differ from DNA or RNA molecules, andanti-sense molecules. Derivative molecules include peptide nucleic acids(PNAs), and non-nucleic acid molecules including phosphorothioate,phosphotriester, phosphoramidate, and methylphosphonate molecules, thatbind to single-stranded DNA or RNA in a base pair-dependent manner(Zamecnik, P. C., et al., 1978 Proc. Natl. Acad. Sci. 75:280284;Goodchild, P. C., et al., 1986 Proc. Natl. Acad. Sci. 83:4143-4146).Peptide nucleic acid molecules comprise a nucleic acid oligomer to whichan amino acid residue, such as lysine, and an amino group have beenadded. These small molecules, also designated anti-gene agents, stoptranscript elongation by binding to their complementary (template)strand of nucleic acid (Nielsen, P. E., et al., 1993 Anticancer Drug Des8:53-63). Reviews of methods for synthesis of DNA, RNA, and theiranalogues can be found in: Oligonucleotides and Analogues, eds. F.Eckstein, 1991, IRL Press, New York; Oligonucleotide Synthesis, ed. M.J. Gait, 1984, IRL Press, Oxford, England. Additionally, methods forantisense RNA technology are described in U.S. Pat. Nos. 5,194,428 and5,110,802. A skilled artisan can readily obtain these classes of nucleicacid molecules using the herein described soluble CTLA4 polynucleotidesequences, see for example Innovative and Perspectives in Solid PhaseSynthesis (1992) Egholm, et al. pp 325-328 or U.S. Pat. No. 5,539,082.

Additionally, the invention provides a vector, which comprises thenucleotide sequences of the invention. The term vector includes, but isnot limited to, plasmids, cosmids, and phagemids. In one embodiment, thevector can be an autonomously replicating vector comprising a repliconthat directs the replication of the rDNA within the appropriate hostcell. Alternatively, the vector can direct integration of therecombinant vector into the host cell. Various viral vectors may also beused, such as, for example, a number of well known retroviral andadenoviral vectors (Berkner 1988 Biotechniques 6:616-629).

The vectors can permit expression of the soluble CTLA4 transcript orpolypeptide sequences in prokaryotic or eukaryotic host cells. Thevectors include expression vectors, comprising an expression controlelement, such as a promoter sequence, which enables transcription of theinserted soluble CTLA4 nucleic acid sequences and can be used forregulating the expression (e.g., transcription and/or translation) of anoperably linked soluble CTLA4 sequence in an appropriate host cell.Expression control elements are known in the art and include, but arenot limited to, inducible promoters, constitutive promoters, secretionsignals, enhancers, transcription terminators, and other transcriptionalregulatory elements. Other expression control elements that are involvedin translation are known in the art, and include the Shine-Dalgarnosequence (e.g., prokaryotic host cells), and initiation and terminationcodons.

Specific initiation signals may also be required for efficienttranslation of a soluble CTLA4 sequence. These signals include theATG-initiation codon and adjacent sequences. In cases where the solubleCTLA4 initiation codon and upstream sequences are inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only the coding sequence,or a portion thereof, is inserted, exogenous transcriptional controlsignals including the ATG-initiation codon may be provided. Furthermore,the initiation codon should be in the correct reading-frame to ensuretranslation of the entire insert. Exogenous transcriptional elements andinitiation codons can be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate to the cell system in use (Scharf, D., et al, 1994Results Probl. Cell. Differ. 20:125-62; Bittner, et al., 1987 Methods inEnzymol. 153:516-544).

The preferred vectors for expression of the soluble CTLA4 sequences ineukaryote host cells include expression control elements, such as thebaculovirus polyhedrin promoter for expression in insect cells. Otherexpression control elements include promoters or enhancers derived fromthe genomes of plant cells (e.g., heat shock, RUBISCO, storage proteingenes), viral promoters or leader sequences or from plant viruses, andpromoters or enhancers from the mammalian genes or from mammalianviruses.

The preferred vector includes at least one selectable marker gene thatencodes a gene product that confers drug resistance such as resistanceto ampicillin or tetracyline. The vector also comprises multipleendonuclease restriction sites that enable convenient insertion ofexogenous DNA sequences. Methods for generating a recombinant expressionvector encoding the soluble CTLA4 proteins of the invention are wellknown in the art, and can be found in Sambrook et al., (MolecularCloning; A Laboratory Manual, 2^(nd) edition, Sambrook, Fritch, andManiatis 1989, Cold Spring Harbor Press) and Ausubel et al. (1989Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y.).

The preferred vectors for generating soluble CTLA4 transcripts and/orthe encoded soluble CTLA4 polypeptides are expression vectors which arecompatible with prokaryotic host cells. Prokaryotic cell expressionvectors are well known in the art and are available from severalcommercial sources. For example, pET vectors (e.g., pET-21, NovagenCorp.), BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.), pSPORT (GibcoBRL, Rockville, Md.), or ptrp-lac hybrids may be used to express solubleCTLA4 polypeptides in bacterial host cells.

Alternatively, the preferred expression vectors for generating solubleCTLA4 transcripts and/or the encoded soluble CTLA4 polypeptides areexpression vectors which are compatible with eukaryotic host cells. Themore preferred vectors are those compatible with vertebrate cells.Eukaryotic cell expression vectors are well known in the art and areavailable from several commercial sources. Typically, such vectors areprovided containing convenient restriction sites for insertion of thedesired DNA segment. Typical of such vectors are PSVL and pKSV-10(Pharmacia), pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1(ATCC, #31255), and similar eukaryotic expression vectors.

Examples of expression vectors for include, but are not limited to,vectors for mammalian host cells (e.g., BPV-1, pHyg, pRSV, pSV2, pTK2(Maniatis); pIRES (Clontech); pRc/CMV2, pRc/RSV, pSFV1 (LifeTechnologies); pVPakc Vectors, pCMV vectors, pSG5 vectors (Stratagene)),retroviral vectors (e.g., pFB vectors (Stratagene)), pcDNA-3(Invitrogen) or modified forms thereof, adenoviral vectors;adeno-associated virus vectors, baculovirus vectors, yeast vectors(e.g., pESC vectors (Stratagene)).

A host vector system is also provided. The host vector system comprisesthe vector of the invention in a suitable host cell. Examples ofsuitable host cells include, but are not limited to, prokaryotic andeukaryotic cells. In accordance with the practice of the invention,eukaryotic cells are also suitable host cells. Examples of eukaryoticcells include any animal cell, whether primary or immortalized, yeast(e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichiapastoris), and plant cells. Exemplary animal cells include cells frombovine, ovine, porcine, murine, equine, monkey and ape. Myeloma, COS andCHO cells are examples of animal cells that may be used as hosts.Particular CHO cells include, but are not limited to, DG44 (Chasin, etla., 1986 Som. Cell. Molec. Genet. 12:555-556; Kolkekar 1997Biochemistry 36:10901-10909), CHO-K1 (ATCC No. CCL-61), CHO-K1 Tet-Oncell line (Clontech), CHO designated ECACC 85050302 (CAMR, Salisbury,Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG,Genova, IT), CHO-K1/SF designated ECACC 93061607 (CAMR, Salisbury,Wiltshire, UK), and RR-CHOK1 designated ECACC 92052129 (CAMR, Salisbury,Wiltshire, UK). Exemplary plant cells include whole plants, cellculture, or callus, from tobacco, corn, soybean, and rice cells. Corn,soybean, and rice seeds are also acceptable.

The CTLA4 mutant molecules of the invention may be isolated asnaturally-occurring polypeptides, or from any source whether natural,synthetic, semi-synthetic or recombinant. Accordingly, the CTLA4 mutantpolypeptide molecules may be isolated as naturally-occurring proteinsfrom any species, particularly mammalian, including bovine, ovine,porcine, murine, equine, and preferably human. Alternatively, the CTLA4mutant polypeptide molecules may be isolated as recombinant polypeptidesthat are expressed in prokaryote or eukaryote host cells, or isolated asa chemically synthesized polypeptide.

A skilled artisan can readily employ standard isolation methods toobtain isolated CTLA4 mutant molecules. The nature and degree ofisolation will depend on the source and the intended use of the isolatedmolecules.

CTLA4 mutant molecules and fragments or derivatives thereof, can beproduced by recombinant methods. Accordingly, an isolated nucleotidesequence encoding wild-type CTLA4 molecules may be manipulated tointroduce mutations, resulting in nucleotide sequences that encode theCTLA4 mutant polypeptide molecules. For example, the nucleotidesequences encoding the CTLA4 mutant molecules may be generated bysite-directed mutagenesis methods, using primers and PCR amplification.The primers can include specific sequences designed to introduce desiredmutations. Alternatively, the primers can be designed to includerandomized or semi-randomized sequences to introduce random mutations.Standard recombinant methods (Molecular Cloning; A Laboratory Manual,2^(nd) edition, Sambrook, Fritch, and Maniatis 1989, Cold Spring HarborPress) and PCR technology (U.S. Pat. No. 4,603,102) can be employed forgenerating and isolating CTLA4 mutant polynucleotides encoding CTLA4mutant polypeptides.

The invention includes pharmaceutical compositions comprisingpharmaceutically effective amounts of a molecule that blocks B7interaction with CTLA4 and/or CD28 such as soluble CTLA4 molecules, CD28molecules, B7 (B7-1 or B7-2) molecules, anti-CTLA4 monoclonalantibodies, anti-CD28 monoclonal antibodies or anti-B7 (B7-1 or B7-2)monoclonal antibodies. The pharmaceutical compositions of the inventionare useful for treatment of cardiovascular diseases. In certainembodiments, cardiovascular diseases are mediated by CD28/CTLA4/B7interactions. The soluble CTLA4 molecules are preferably soluble CTLA4molecules with wildtype sequence and/or soluble CTLA4 molecules havingone or more mutations in the extracellular domain of CTLA4. Thepharmaceutical composition can include soluble CTLA4 protein moleculesand/or nucleic acid molecules, and/or vectors encoding the molecules. Inpreferred embodiments, the soluble CTLA4 molecules have the amino acidsequence of the extracellular domain of CTLA4 as shown in either FIG. 24or 19 (CTLA4Ig or L104EA29Y, respectively). Even more preferably, thesoluble CTLA4 mutant molecule is L104EA29YIg as disclosed herein.

The compositions of the invention may additionally include one or moreadditional, other, or second therapeutic agents. By “administered incombination” or “combination therapy” it is meant that a compound of thepresent invention and one or more additional therapeutic agents are bothadministered to the mammal being treated. When administered incombination each component may be administered at the same time orsequentially in any order at different points in time. Thus, eachcomponent may be administered separately but sufficiently closely intime so as to provide the desired therapeutic effect.

Additional therapeutic agents, when employed in combination with themolecules of the present invention, may be used, for example, in thoseamounts indicated in the Physicians' Desk Reference (PDR) or asotherwise determined by one of ordinary skill in the art.

Additional, other, or second therapeutic agents include anti-coagulantor coagulation inhibitory agents, anti-platelet or platelet inhibitoryagents, thrombin inhibitors, Vitamin K antagonists, glycoproteinIIb/IIIa receptor antagonists, thrombolytic or fibrinolytic agents,anti-arrythmic agents, anti-hypertensive agents, angiotensin convertingenzyme inhibitors (ACE-Is), angiotensin receptor blockers (ARBs),beta-blockers, calcium channel blockers (L-type and T-type), cardiacglycosides, diruetics, mineralocorticoid receptor antagonists,phospodiesterase inhibitors, cholesterol/lipid lowering agents and lipidprofile therapies, anti-diabetic agents, anti-depressants,anti-inflammatory agents (steroidal and non-steroidal),anti-osteoporosis agents, hormone replacement therapies, oralcontraceptives, anti-obesity agents, anti-anxiety agents,anti-proliferative agents, anti-tumor agents, anti-ulcer andgastroesophageal reflux disease agents, growth hormone and/or growthhormone secretagogues, thyroid mimetics (including thyroid receptorantagonist), anti-infective agents, anti-viral agents, anti-bacterialagents, and anti-fungal agents.

Anticoagulant agents (or coagulation inhibitory agents) that may be usedin combination with the compositions of this invention include warfarinand heparin (either unfractionated heparin or any commercially availablelow molecular weight heparin), synthetic pentasaccharide, direct actingthrombin inhibitors including hirudin and argatroban as well as factorXa inhibitors.

The term anti-platelet agents (or platelet inhibitory agents), as usedherein, denotes agents that inhibit platelet function, for example byinhibiting the aggregation, adhesion or granular secretion of platelets.Agents include, but are not limited to, the various known non-steroidalanti-inflammatory drugs (NSAIDS) such as aspirin, ibuprofen, naproxen,sulindac, indomethacin, mefenamate, droxicam, diclofenac,sulfinpyrazone, piroxicam, and pharmaceutically acceptable salts orprodrugs thereof. Of the NSAIDS, aspirin (acetylsalicyclic acid or ASA)and piroxicam are preferred. Other suitable platelet inhibitory agentsinclude IIb/IIIa antagonists (e.g., tirofiban, eptifibatide, andabciximab), thromboxane-A2-receptor antagonists (e.g., ifetroban),thromboxane-A2-synthetase inhibitors, PDE-III inhibitors (e.g.,dipyridamole), and pharmaceutically acceptable salts or prodrugsthereof.

The term anti-platelet agents (or platelet inhibitory agents), as usedherein, is also intended to include ADP (adenosine diphosphate) receptorantagonists, preferably antagonists of the purinergic receptors P₂Y₁ andP₂Y₁₂, with P₂Y₁₂ being even more preferred. Preferred P₂Y₁₂ receptorantagonists include ticlopidine and clopidogrel, includingpharmaceutically acceptable salts or prodrugs thereof. Clopidogrel is aneven more preferred agent. Ticlopidine and clopidogrel are alsopreferred compounds since they are known to be gentle on thegastro-intestinal tract in use.

The term thrombin inhibitors (or anti-thrombin agents), as used herein,denotes inhibitors of the serine protease thrombin. By inhibitingthrombin, various thrombin-mediated processes, such as thrombin-mediatedplatelet activation (that is, for example, the aggregation of platelets,and/or the granular secretion of plasminogen activator inhibitor-1and/or serotonin) and/or fibrin formation are disrupted. A number ofthrombin inhibitors are known to one of skill in the art and theseinhibitors are contemplated to be used in combination with the presentcompounds. Such inhibitors include, but are not limited to, boroargininederivatives, boropeptides, heparins, hirudin, argatroban, andmelagatran, including pharmaceutically acceptable salts and prodrugsthereof. Boroarginine derivatives and boropeptides include N-acetyl andpeptide derivatives of boronic acid, such as C-terminal α-aminoboronicacid derivatives of lysine, omithine, arginine, homoarginine andcorresponding isothiouronium analogs thereof. The term hirudin, as usedherein, includes suitable derivatives or analogs of hirudin, referred toherein as hirulogs, such as disulfatohirudin. The term thrombolytics orfibrinolytic agents (or thrombolytics or fibrinolytics), as used herein,denote agents that lyse blood clots (thrombi). Such agents includetissue plasminogen activator (natural or recombinant) and modified formsthereof, anistreplase, urokinase, streptokinase, tenecteplase (TNK),lanoteplase (nPA), factor VIIa inhibitors, PAI-1 inhibitors (i.e.,inactivators of tissue plasminogen activator inhibitors),alpha2-antiplasmin inhibitors, and anisoylated plasminogen streptokinaseactivator complex, including pharmaceutically acceptable salts orprodrugs thereof. The term anistreplase, as used herein, refers toanisoylated plasminogen streptokinase activator complex, as described,for example, in EP 028,489, the disclosure of which is herebyincorporated herein by reference herein. The term urokinase, as usedherein, is intended to denote both dual and single chain urokinase, thelatter also being referred to herein as prourokinase.

Examples of suitable anti-arrythmic agents for use in combination withthe present compounds include: Class I agents (such as propafenone);Class II agents (such as carvadiol and propranolol); Class III agents(such as sotalol, dofetilide, amiodarone, azimilide and ibutilide);Class IV agents (such as ditiazem and verapamil); K⁺ channel openerssuch as I_(Ach) inhibitors, and I_(Kur) inhibitors (e.g., compounds suchas those disclosed in WO01/40231).

Examples of suitable anti-hypertensive agents for use in combinationwith the compounds of the present invention include: alpha adrenergicblockers; beta adrenergic blockers; calcium channel blockers (e.g.,diltiazem, verapamil, nifedipine, amlodipine and mybefradil); diruetics(e.g., chlorothiazide, hydrochlorothiazide, flumethiazide,hydroflumethiazide, bendroflumethiazide, methylchlorothiazide,trichloromethiazide, polythiazide, benzthiazide, ethacrynic acidtricrynafen, chlorthalidone, furosemide, musolimine, bumetamide,triamtrenene, amiloride, spironolactone); renin inhibitors; ACEinhibitors (e.g., captopril, zofenopril, fosinopril, enalapril,ceranopril, cilazopril, delapril, pentopril, quinapril, ramipril,lisinopril); AT-1 receptor antagonists (e.g., losartan, irbesartan,valsartan); ET receptor antagonists (e.g., sitaxsentan, atrsentan andcompounds disclosed in U.S. Pat. Nos. 5,612,359 and 6,043,265); DualET/AII antagonist (e.g., compounds disclosed in WO 00/01389); neutralendopeptidase (NEP) inhibitors; vasopepsidase inhibitors (dual NEP-ACEinhibitors) (e.g., omapatrilat, gemopatrilat and nitrates).

Examples of suitable calcium channel blockers (L-type or T-type) for usein combination with the compounds of the present invention includediltiazem, verapamil, nifedipine, amlodipine and mybefradil.

Examples of suitable cardiac glycosides for use in combination with thecompounds of the present invention include digitalis and ouabain.

Examples of suitable diruetics for use in combination with the compoundsof the present invention include: chlorothiazide, hydrochlorothiazide,flumethiazide, hydroflumethiazide, bendroflumethiazide,methylchlorothiazide, trichloromethiazide, polythiazide, benzthiazide,ethacrynic acid tricrynafen, chlorthalidone, furosemide, musolimine,bumetanide, triamtrenene, amiloride, and spironolactone.

Examples of suitable mineralocorticoid receptor antagonists for use incombination with the compounds of the present invention includesprionolactone and eplerenone.

Examples of suitable phospodiesterase inhibitors for use in combinationwith the compounds of the present invention include: PDE III inhibitors(such as cilostazol); and PDE V inhibitors (such as sildenafil).

Examples of suitable cholesterol/lipid lowering agents and lipid profiletherapies for use in combination with the compounds of the presentinvention include: HMG-CoA reductase inhibitors (e.g., pravastatin,lovastatin, atorvastatin, simvastatin, fluvastatin, NK-104 (a.k.a.itavastatin, or nisvastatin or nisbastatin) and ZD-4522 (a.k.a.rosuvastatin, or atavastatin or visastatin)); squalene synthetaseinhibitors; fibrates; bile acid sequestrants (such as questran); ACATinhibitors; MTP inhibitors; lipooxygenase inhibitors; choesterolabsorption inhibitors; and cholesterol ester transfer protein inhibitors(e.g., CP-529414).

Examples of suitable anti-diabetic agents for use in combination withthe compounds of the present invention include: biguanides (e.g.,metformin); glucosidase inhibitors (e.g., acarbose); insulins (includinginsulin secretagogues or insulin sensitizers); meglitinides (e.g.,repaglinide); sulfonylureas (e.g., glimepiride, glyburide andglipizide); biguanide/glyburide combinations (e.g., glucovance),thiozolidinediones (e.g., troglitazone, rosiglitazone and pioglitazone),PPAR-alpha agonists, PPAR-gamma agonists, PPAR alpha/gamma dualagonists, SGLT2 inhibitors, inhibitors of fatty acid binding protein(aP2) such as those disclosed in WO00/59506, glucagon-like peptide-1(GLP-1), and dipeptidyl peptidase IV (DP4) inhibitors.

Examples of suitable anti-depressant agents for use in combination withthe compounds of the present invention include nefazodone andsertraline.

Examples of suitable anti-inflammatory agents for use in combinationwith the compounds of the present invention include: steroid compoundssuch as corticosteroids and glucocorticoids, including prednisone anddexamethasone; etanercept (Enbrel®); infliximab (Remicade®); proteintyrosine kinase (PTK) inhibitors; cyclooxygenase inhibitors (includingNSAIDs, and COX-1 and/or COX-2 inhibitors); aspirin; indomethacin;ibuprofen; prioxicam; naproxen; celecoxib; and/or rofecoxib.

Examples of suitable anti-osteoporosis agents for use in combinationwith the compounds of the present invention include alendronate andraloxifene.

Examples of suitable hormone replacement therapies for use incombination with the compounds of the present invention include estrogen(e.g., congugated estrogens) and estradiol.

Examples of suitable anti-coagulants for use in combination with thecompounds of the present invention include heparins (e.g., unfractionedand low molecular weight heparins such as enoxaparin and dalteparin).

Examples of suitable anti-obesity agents for use in combination with thecompounds of the present invention include orlistat and aP2 inhibitors(such as those disclosed in WO00/59506).

Examples of suitable anti-anxiety agents for use in combination with thecompounds of the present invention include diazepam, lorazepam,buspirone, and hydroxyzine pamoate.

Examples of suitable anti-proliferative agents for use in combinationwith the compounds of the present invention include cyclosporin A,paclitaxel, adriamycin; epithilones, cisplatin, and carboplatin.

Examples of suitable anti-ulcer and gastroesophageal reflux diseaseagents for use in combination with the compounds of the presentinvention include famotidine, ranitidine, and omeprazole.

Additional therapeutic agents also include: collagen, dnaj, moleculesthat block TNF function (e.g., pegsunercept), molecules that blockcytokine function (e.g., AMG719), molecules that block LFA-1 function(e.g., efalizumab) and stem cell transplants. These other treatments arecurrently being studied in clinical trials (www.clinicaltrials.gov) todetermine their effect on rheumatoid arthritis.

Collagen, for example in the form of bovine II collagen, may be orallyadministered to a patient suffering from cardiovascular disease in orderto alleviate one or more symptoms of cardiovascular disease.

DnaJ is a small peptide which mimics a protein contained in a gene inmany patients with cardiovascular disease. The peptide is derived fromE. coli bacteria heat shock protein. DnaJ may be orally administered toa patient suffering from cardiovascular disease in order to alleviateone or more symptoms of cardiovascular disease.

TNF is a molecule involved in the inflammatory response of patients withrheumatoid arthritis and possibly cardiovascular disease. Conceivably,any molecule that blocks TNF function e.g., by blocking TNF binding tothe TNF receptor (TNFR), may help modify the progression ofcardiovascular disease and alleviate some of its symptoms. Several TNFblockers such as infliximab and etanercept, have been shown to beefficacious in treating cardiovascular disease. Other TNF blockers suchas pegsunercept are being developed and tested (Phase II clinical trial)for their efficacy in treating cardiovascular disease.

Cytokines e.g., Interleukin-1 (IL-1), are cell secreted moleculesinvolved in mediating immune responses. Conceivably, any molecule thatblocks cytokine function e.g., by blocking IL-1 interaction with itsreceptor, may help modify the progression of cardiovascular disease andalleviate one or more of its symptoms. Anakinra, a recombinant proteinthat blocks IL-1 interaction with its receptor (IL-1 R) has been shownto be efficacious in treating cardiovascularoid arthritis. An IL-1inhibitor, AMG719, is being developed and tested (Phase II clinicaltrial) for its efficacy in treating rheumatoid arthritis.

Lymphocyte function associated molecule 1 (LFA-1) is a molecule composedof two subunits, CD11a and CD18, which functions by mediating lymphocyteadhesion to various cell types such as endothelium. Conceivably,interference of LFA-1 function may help modify the progression ofcardiovascular disease and alleviate one or more of its symptoms. Ananti-LFA-1 antibody, efalizumab, is being developed and tested (Phase IIclinical trial) for its efficacy in treating rheumatoid arthritis.

Blockage of TNF, cytokine or LFA-1 interaction to their ligands by apotentially therapeutic molecule can be determined by any number ofassays known to those skilled in the art. For example, competitionassays may be used to test blockage by the molecule of interest e.g., amolecule can be exposed to a TNF/TNFR binding pair in order to competewith TNF to bind to TNFR. Alternatively, functional assays can beperformed to test blockage e.g., a molecule can be tested for itsability to inhibit an inflammatory cascade, or any part of aninflammatory reaction such as swelling, redness or pain, caused by acytokine.

Additional therapeutic agents also include: p38 MAP kinase inhibitor,soluble gp39 (also known as CD40 ligand (CD40L), CD154, T-BAM, TRAP),soluble CD29, soluble CD40, soluble CD80 (e.g. ATCC 68627), solubleCD86, soluble CD28 (e.g. ATCC accession number 68628), soluble CD56,soluble Thy-1, soluble CD3, soluble TCR, soluble VLA-4, soluble VCAM-1,soluble LECAM-1, soluble ELAM-1, soluble CD44, antibodies reactive withgp39 (e.g. ATCC HB-10916, ATCC HB-12055 and ATCC HB-12056), antibodiesreactive with CD40 (e.g. ATCC HB-9110), antibodies reactive with B7(e.g. ATCC HB-253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB-301, ATCCHB-11341, etc), antibodies reactive with CD28 (e.g. ATCC HB-11944 or mAb9.3 as described by Martin et al (J. Clin. Immun. 4(1):18-22, 1980),antibodies reactive with LFA-1 (e.g. ATCC HB-9579 and ATCC TIB-213),antibodies reactive with LFA-2, antibodies reactive with IL-2,antibodies reactive with IL-12, antibodies reactive with IFN-gamma,antibodies reactive with CD2, antibodies reactive with CD48, antibodiesreactive with any ICAM (e.g., ICAM-1 (ATCC CRL-2252), ICAM-2 andICAM-3), antibodies reactive with CTLA4 (e.g. ATCC HB-304), antibodiesreactive with Thy-1, antibodies reactive with CD56, antibodies reactivewith CD3, antibodies reactive with CD29, antibodies reactive with TCR,antibodies reactive with VLA-4, antibodies reactive with VCAM-1,antibodies reactive with LECAM-1, antibodies reactive with ELAM-1,antibodies reactive with CD44. In certain embodiments, monoclonalantibodies are preferred. In other embodiments, antibody fragments arepreferred. As persons skilled in the art will readily understand, thecombination can include: the soluble CTLA4 molecules of the inventionand one other agent; the soluble CTLA4 molecules with two other agents;the soluble CTLA4 molecules with three other agents; and the like. Thedetermination of the optimal combination and dosages can be determinedand optimized using methods well known in the art.

Some specific combinations for co-administration include the following:CTLA4Ig or L104EA29YIg and CD80 monoclonal antibodies (mAbs); CTLA4Ig orL104EA29YIg and CD86 mAbs; CTLA4Ig or L104EA29YIg, CD80 mAbs, and CD86mAbs; CTLA4Ig or L104EA29YIg and gp39 mAbs; CTLA4Ig or L104EA29YIg andCD40 mAbs; CTLA4Ig or L104EA29YIg and CD28 mAbs; CTLA4Ig or L104EA29YIg,CD80 and CD86 mAbs, and gp39 mAbs; CTLA4Ig or L104EA29YIg, CD80 and CD86mAbs and CD40 mAbs; and CTLA4Ig or L104EA29YIg, anti-LFA1 mAb, andanti-gp39 mAb. A specific example of a gp39 mAb is MR1. Othercombinations will be readily appreciated and understood by personsskilled in the art.

Additional therapeutic agents also include: a calcineurin inhibitor,e.g. cyclosporin A or FK506; an immunosuppressive macrolide, e.g.rapamycine or a derivative thereof (e.g.40-O-(2-hydroxy)ethyl-rapamycin); a lymphocyte homing agent, e.g. FTY720or an analog thereof; corticosteroids; cyclophosphamide; azathioprene; adihydrofolic acid reductase inhibitor such as methotrexate; leflunomideor an analog thereof; mizoribine; mycophenylic acid; mycophenylatemofetil; 15-deoxyspergualine or an analog thereof; immunosuppressivemonoclonal antibodies, e.g., monoclonal antibodies to leukocytereceptors, e.g., MHC, CD2, CD3, CD4, CD 11a/CD18, CD7, CD25, CD 27, B7,CD40, CD45, CD58, CD137, ICOS, CD150 (SLAM), OX40, 4-1BB or theirligands; or other immunomodulatory compounds, e.g. CTLA4/CD28-Ig, orother adhesion molecule inhibitors, e.g. mAbs or low molecular weightinhibitors including LFA-1 antagonists, Selectin antagonists and VLA-4antagonists. The compound is particularly useful in combination with acompound that interferes with CD40 and its ligand, e.g. antibodies toCD40 and antibodies to CD40-L.

Administration of the compounds of the present invention (i.e., a firsttherapeutic agent) in combination with at least one additionaltherapeutic agent (i.e., a second therapeutic agent), preferably affordsan efficacy advantage over the compounds and agents alone, preferablywhile permitting the use of lower doses of each (i.e., a synergisticcombination). A lower dosage minimizes the potential of side effects,thereby providing an increased margin of safety. It is preferred that atleast one of the therapeutic agents is administered in a sub-therapeuticdose. It is even more preferred that all of the therapeutic agents beadministered in sub-therapeutic doses. Sub-therapeutic is intended tomean an amount of a therapeutic agent that by itself does not give thedesired therapeutic effect for the condition or disease being treated.Synergistic combination is intended to mean that the observed effect ofthe combination is greater than the sum of the individual agentsadministered alone.

A pharmaceutical composition comprising soluble CTLA4 can be used formethods for blocking B7 interaction with CTLA4 and/or CD28; or fortreating cardiovascular diseases. Effective amounts of soluble CTLA4 inthe pharmaceutical composition range about 0.1 to 100 mg/kg weight ofthe subject. In another embodiment, the effective amount is an amountabout 0.5 to 100 mg/kg weight of a subject, 0.5 to 5 mg/kg weight of asubject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kgweight of a subject, about 15 to 20 mg/kg weight of a subject, about 20to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of asubject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kgweight of a subject, about 40 to 45 mg/kg of a subject, about 45 to 50mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject,about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight ofa subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject,about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight ofa subject, about 90 to 95 mg/kg weight of a subject, or about 95 to 100mg/kg weight of a subject.

In an embodiment, the effective amount of soluble CTLA4 is an amountabout 2 mg/kg to about 10 mg/kg weight of a subject. In anotherembodiment, the effective amount is an amount about 0.1 to 4 mg/kgweight of a subject. In another embodiment the effective amount is anamount about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject,about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kgweight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about3.0 to 3.5 mg/kg weight of a subject or about 3.5 to 4.0 mg/kg weight ofa subject. In another embodiment, the effective amount is an amountabout 0.1 to 20 mg/kg weight of a subject. In another embodiment, theeffective amount is an amount about 0.1 to 2 mg/kg weight of a subject,about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of asubject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kgweight of a subject, about 10 to 12 mg/kg weight of a subject, about 12to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of asubject, about 16 to 18 mg/kg weight of a subject or about 18 to 20mg/kg weight of a subject. In an embodiment, the effective amount is 2mg/kg weight of a subject. In another embodiment, the effective amountis about 10 mg/kg weight of a subject.

In a specific embodiment, an effective amount of soluble CTLA4 is 500 mgfor a subject weighing less than 60 kg, 750 mg for a subject weighingbetween 60-100 kg and 1000 mg for a subject weighing more than 100 kg.

The present invention also provides pharmaceutical compositionscomprising the molecules of the inventon e.g., CTLA4Ig and an acceptablecarrier or adjuvant which is known to those of skill of the art. Thepharmaceutical compositions preferably include suitable carriers andadjuvants which include any material which when combined with themolecules of the invention (e.g., a soluble CTLA4 molecule, such as,CTLA4Ig or L104EA29Y) retain the molecule's activity, and isnon-reactive with the subject's immune system. These carriers andadjuvants include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, phosphate buffered saline solution, water, emulsions (e.g.oil/water emulsion), salts or electrolytes such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances and polyethylene glycol. Othercarriers may also include sterile solutions; tablets, including coatedtablets and capsules. Typically such carriers contain excipients such asstarch, milk, sugar (e.g. sucrose, glucose, maltose), certain types ofclay, gelatin, stearic acid or salts thereof, magnesium or calciumstearate, talc, vegetable fats or oils, gums, glycols, or other knownexcipients. Such carriers may also include flavor and color additives orother ingredients. Compositions comprising such carriers are formulatedby well known conventional methods. Such compositions may also beformulated within various lipid compositions, such as, for example,liposomes as well as in various polymeric compositions, such as polymermicrospheres.

In a further embodiment of the invention, the present invention provideskits (i.e., a packaged combination of reagents with instructions)containing the molecules of the invention useful for blocking B7interactions with its ligands and/or for treating a cardiovasculardisease.

The kit can contain a pharmaceutical composition that includes one ormore agents, for example, a soluble CTLA4 molecule alone, or with asecond agent, and an acceptable carrier or adjuvant, e.g.,pharmaceutically acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use. The agents may be provided as dry powders,usually lyophilized, including excipients that upon dissolving willprovide a reagent solution having the appropriate concentration.

Second agents can include those described above as additional agents,such as anti-coagulant or coagulation inhibitory agents, anti-plateletor platelet inhibitory agents, thrombin inhibitors, thrombolytic orfibrinolytic agents, anti-arrythmic agents, anti-hypertensive agents,calcium channel blockers (L-type and T-type), cardiac glycosides,diruetics, mineralocorticoid receptor antagonists, phospodiesteraseinhibitors, cholesterol/lipid lowering agents and lipid profiletherapies, anti-diabetic agents, anti-depressants, anti-inflammatoryagents (steroidal and non-steroidal), anti-osteoporosis agents, hormonereplacement therapies, oral contraceptives, anti-obesity agents,anti-anxiety agents, anti-proliferative agents, anti-tumor agents,anti-ulcer and gastroesophageal reflux disease agents, growth hormoneand/or growth hormone secretagogues, thyroid mimetics (including thyroidreceptor antagonist), anti-infective agents, anti-viral agents,anti-bacterial agents, and anti-fungal agents.

The kit comprises a container with a label and/or instructions. Suitablecontainers include, for example, bottles, vials, and test tubes. Thecontainers can be formed from a variety of materials such as glass orplastic. The container can have a sterile access port (for example thecontainer can be an intravenous solution bag or a vial having a stopperpierceable by a needle such as a hypodermic injection needle). Thecontainer can hold a pharmaceutical composition such as a pharmaceuticalcomposition having an agent that is effective for blocking B7interactions with its ligand and/or treating an cardiovascular disease.

The kit can also comprise a second container comprising one or moresecond agents as described herein and/or a pharmaceutically acceptablebuffer, such as phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

The kit may also suitably include a label and/or instructions on, orassociated with the container. The label can provide directions forcarrying out the preparation of the agents for example, dissolving ofthe dry powders, and/or treatment for a specific cardiovascular disease.The label and/or instructions may indicate that administration of thecomposition of the invention and a second agent may be at the same timeor may be sequentially in any order at different points in time.

The label and/or the instructions can indicate directions for either invivo or in vitro use of the pharmaceutical composition. The label and/orthe instructions can indicate that the pharmaceutical composition isused alone, or in combination with a second agent.

The label can indicate appropriate dosages for the molecules of theinvention. For example, the label can indicate that dosages for amolecule that is effective for blocking B7 interactions with its ligandand/or treating an cardiovascular disease is about 0.1 to 100 mg/kgweight of the subject, about 0.5 to 100 mg/kg weight of a subject, 0.5to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject,about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight ofa subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject,about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of asubject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kgweight of a subject, about 55 to 60 mg/kg weight of a subject, about 60to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of asubject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kgweight of a subject, about 80 to 85 mg/kg weight of a subject, about 85to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of asubject, about 95 to 100 mg/kg weight of a subject, about 2 to 10 mg/kgweight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of asubject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject,about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kgweight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of asubject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject,about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kgweight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of asubject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject,about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of asubject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kgweight of a subject, about 10 to 12 mg/kg weight of a subject, about 12to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of asubject, about 16 to 18 mg/kg weight of a subject, about 18 to 20 mg/kgweight of a subject, about 0.5 mg/kg weight of the subject, 2 mg/kgweight of the subject, 10 mg/kg weight of the subject, about 0.5 mg/kgto 100 weight of the subject, about 0.5 to 10 mg/kg weight of a subject,about 0.1 to 20 mg/kg weight of a subject, about 500 mg for a subjectweighing less than 60 kg, 750 mg for a subject weighing between 60-100kg or 1000 mg for a subject weighing more than 100 kg.

The label and/or the instructions can also indicate that thepharmaceutical composition can be used alone, or in combination, with asecond agent to treat a condition of choice, e.g., cardiovasculardiseases, at the same time or sequentially in any order at differentpoints in time.

In a specific embodiment of the invention, the kit comprises apharmaceutical composition comprising a pharmaceutically acceptablecarrier and an effective amount of a first agent, wherein the firstagent is a molecule that blocks B7 interaction with CTLA4 and/or CD28such as soluble CTLA4 molecules, CD28 molecules, B7 (B7-1 or B7-2)molecules, anti-CTLA4 monoclonal antibodies, anti-CD28 monoclonalantibodies or anti-B7 (B7-1 or B7-2) monoclonal antibodies. In preferredembodiments, the soluble CTLA4 molecules have the amino acid sequence ofthe extracellular domain of CTLA4 as shown in either FIG. 24 or 19(CTLA4Ig or L104EA29Y, respectively).

Methods

The invention provides methods for regulating functional CTLA4- andCD28-positive cell interactions with B7-positive cells. The methodscomprise contacting the B7-positive cells with a soluble CTLA4 moleculeof the invention so as to regulate functional CTLA4- and CD28-positivecell interactions with B7-positive cells, e.g., by interfering withreaction of an endogenous CTLA4 and/or CD28 molecule with a B7 molecule.Suitable amounts of soluble CTLA4 for use in the methods of theinvention are described supra.

The present invention further provides methods for treatingcardiovascular diseases. The methods comprise administering atherapeutic composition of the invention, such as soluble CTLA4molecules of the invention, to a subject in an amount effective torelieve at least one of the symptoms associated with cardiovasculardiseases. Examples of soluble CTLA4 include CTLA4Ig and soluble CTLA4mutant molecule e.g. L104EA29YIg. Symptoms of cardiovascular diseaseinclude, but are not limited to, dysrhythmias; ischemia; angina; reducedexercise tolerance; fatigue; dyspnea on exertion; and transient ischemicattacks. Additionally, the invention may provide long-term therapy forcardiovascular diseases by blocking the T-cell/B7-positive cellinteractions, thereby blocking T-cell activation/stimulation byco-stimulatory signals such as B7 binding to CD28, leading to inductionof T-cell anergy or tolerance.

Cardiovascular diseases include, but are not limited to, the followingdiseases or conditions: thromboembolic disorders, including arterialcardiovascular thromboembolic disorders, venous cardiovascularthromboembolic disorders, and thromboembolic disorders in the chambersof the heart; ahtherosclerosis; restensosis; peripheral arterialdisease; coronary bypass grafting surgery; carotid artery disease;arteritis; myocarditis; cardiovascular inflammation; vascularinflammation; coronary heart disease (CHD); unstable angina (UA);unstable refractory angina; stable angina (SA); chronic stable angina;acute coronary syndrome (ACS); first or recurrent myocardial infarction;acute myocardial infarction (AMI); myocardial infarction; non-Q wavemyocardial infarction; non-STE myocardial infarction; coronary arterydisease; cardiac ischemia; ischemia; ischemic sudden death; transientischemic attack; stroke; atherosclerosis; peripheral occlusive arterialdisease; venous thrombosis; deep vein thrombosis; thrombophlebitis;arterial embolism; coronary arterial thrombosis; cerebral arterialthrombosis; cerebral embolism; kidney embolism; pulmonary embolism;thrombosis resulting from (a) prosthetic valves or other implants, (b)indwelling catheters, (c) stents, (d) cardiopulmonary bypass, (e)hemodialysis, or (f) other procedures in which blood is exposed to anartificial surface that promotes thrombosis; thrombosis resulting fromatherosclerosis, surgery or surgical complications, prolongedimmobilization, arterial fibrillation, congenital thrombophilia, cancer,diabetes, effects of medications or hormones, and complications ofpregnancy; cardiac arrhytmias including supraventricular arrhythmias,atrial arrhythmias, atrial flutter, atrial fibrillation; other diseaseslisted in Heart Disease: A Textbook of Cardiovascular Medicine, 2 VolumeSet, 6th Edition, 2001, Eugene Braunwald, Douglas P. Zipes, Peter Libby,Douglas D. Zipes.

Preferred cardiovascular diseases are: atherosclerosis; coronary heartdisease (CHD); restensosis; peripheral arterial disease; coronary bypassgrafting surgery; carotid artery disease; arteritis; myocarditis;cardiovascular inflammation; vascular inflammation; unstable angina(UA); unstable refractory angina; stable angina (SA); chronic stableangina; acute coronary syndrome (ACS); myocardial infarction; acutemyocardial infarction (AMI), including first or recurrent myocardialinfarction, non-Q wave myocardial infarction, non-ST-segment elevationmyocardial infarction and ST-segment elevation myocardial infarction.

More preferred cardiovascular diseases are: atherosclerosis; coronaryheart disease (CHD); unstable angina (UA); unstable refractory angina;stable angina (SA); chronic stable angina; acute coronary syndrome(ACS); myocardial infarction; acute myocardial infarction (AMI),including first or recurrent myocardial infarction, non-Q wavemyocardial infarction, non-ST-segment elevation myocardial infarctionand ST-segment elevation myocardial infarction.

Preferred cardiovascular diseases are carcardiovascular diseasesmediated by T cell interactions with B7-positive cells.

Also preferred are cardiovascular diseases associated with at least onemarker of inflammation. By associated with a marker of inflammation, ismeant that the marker (presence, absence or specific concentrations)correlates statisitically with: the risk of the future development ofdisease, or the risk of disease recurrence, or the current level ofdisease activity. Markers of inflammation include, but are not limitedto, CRP, hsCRP, IL-10, CD40L, sCD40L, IL-6, sICAM-1, TNF-α, white bloodcell count, fibrinogen, and serum amyloid A. Preferred markers ofinflammation are CRP, hsCRP, IL-6, and TNF-α; most preferred is hsCRP.High CRP, high hsCRP, low IL-10, high sCD40L, high IL-6, high sICAM-1,high TNF-α, high white blood cell count, high fibrinogen, and high serumamyloid A are known to be indicators of inflammation.

The soluble CTLA4 molecules of the invention exhibit inhibitoryproperties in vivo. Under conditions where T-cell/B7-positive cellinteractions, for example T cell/B cell interactions, are occurring as aresult of contact between T cells and B7-positive cells, binding ofintroduced CTLA4 molecules to react to B7-positive cells, for example Bcells, may interfere, i.e., inhibit, the T cell/B7-positive cellinteractions resulting in regulation of immune responses.

Preferred embodiments of the invention comprises use of CTLA4Ig or thesoluble CTLA4 mutant molecule L104EA29YIg to regulate functional CTLA4-and CD28-positive cell interactions with B7-positive cells, to treat orprevent cardiovascular diseases, including recurrent cardiovascularevents (e.g., myocardial infarction), and/or to downregulate immuneresponses. The L104EA29YIg of the invention is a soluble CTLA4 mutantmolecule comprising at least the two amino acid changes, the leucine (L)to glutamic acid (E) at position +104 and the alanine (A) to tyrosine(Y) change at position +29 (FIG. 19). The L104EA29YIg molecule mayencompass further mutations beyond the two specified herein.

Suitable amounts of the molecule used to block the B7 interaction withCTLA4 and/or CD28 are described supra. The molecule used to block theB7/CTLA4 interaction may be a soluble CTLA4 such as CTLA4Ig,CTLA4Ig/CD28Ig or L104EA29YIg, a soluble CD28 such as CD28Ig, a solubleB7 (B7-1 or B7-2) such as B7Ig, anti-CTLA4 monoclonal antibodies,anti-CD28 monoclonal antibodies or anti-B7 monoclonal antibodies.

The amount of symptom relief provided by the present invention can bemeasured using any of the accepted criteria established to measure anddocument symptom relief in a clinical setting. Acceptable criteria formeasuring symptom relief may include: angina; recurrent angina; unstableangina; ischemic time; hospitalization for cardiovascular reasons; acutemyocardial infarction; recurrent myocardial infarction; need for urgentrevascularization; need for percutaneous coronary intervention; allcause mortality; cardiovascular mortality; transent ischemic attack; andstroke.

The subjects treated by the present invention include mammaliansubjects, including, human, monkey, ape, dog, cat, cow, horse, goat,pig, rabbit, mouse and rat.

The present invention provides various methods, local or systemic, foradministering the therapeutic compositions of the invention such assoluble CTLA4 molecule alone or in conjunction with an additionaltherapeutic agent. The methods include intravenous, intramuscular,intraperitoneal, oral, inhalation and subcutaneous methods, as well asimplantable pump, continuous infusion, bolus infusion, gene therapy,liposomes, suppositories, topical contact, vesicles, capsules,biodegradable polymers, drug-eluting stents, hydrogels, controlledrelease patch, and injection methods. The therapeutic agent, compoundedwith a carrier, is commonly lyophilized for storage and is reconstitutedwith water or a buffered solution with a neutral pH (about pH 7-8, e.g.,pH 7.5) prior to administration.

The therapeutic composition of the invention, such as soluble CTLA4molecule, may be administered at the same time, or may be administeredsequentially in any order at different points in time, as an additionaltherapeutic agent.

As is standard practice in the art, the compositions of the inventionmay be administered to the subject in any pharmaceutically acceptableform.

In accordance with the practice of the invention, the methods compriseadministering to a subject the soluble CTLA4 molecules of the inventionto regulate CD28- and/or CTLA4-positive cell interactions withB7-positive cells. The B7-positive cells are contacted with an effectiveamount of the soluble CTLA4 molecules of the invention, or fragments orderivatives thereof, so as to form soluble CTLA4/B7 complexes. Suitableamounts of soluble CTLA4 are described supra. The complexes interferewith interaction between endogenous CTLA4 and CD28 molecules with B7family molecules.

The soluble CTLA4 molecules may be administered to a subject in anamount and for a time (e.g. length of time and/or multiple times)sufficient to block endogenous B7 molecules from binding theirrespective ligands, in the subject. Blockage of endogenous B7/ligandbinding thereby inhibiting interactions between B7-positive cells withCD28- and/or CTLA4-positive cells. In an embodiment, soluble CTLA4 maybe administered to a subject daily, weekly, monthly and/or yearly, insingle or multiple times per day/week/month/year, depending on need. Forexample, in one embodiment, the molecule may initially be administeredonce every two weeks for a month, and then once every month thereafter.

Dosage of a therapeutic agent is dependant upon many factors including,but not limited to, the type of tissue affected, the type ofcardiovascular disease being treated, the severity of the disease, asubject's health and response to the treatment with the agents.Accordingly, dosages of the agents can vary depending on each subjectand the mode of administration. The soluble CTLA4 molecules may beadministered in an amount from about 0.1 to 100 mg/kg weight of thepatient/day. Suitable amounts of soluble CTLA4 are described supra.

The invention also encompasses the use of the compositions of theinvention together with other therapeutic agents or pharmaceuticalagents to treat cardiovascular diseases. For example, cardiovasculardiseases may be treated with molecules of the invention in conjunctionwith, but not limited to, the additional therapeutic agents listed abovein the discussion of compositions of the invention.

Where the soluble CTLA4 mutant molecules of the invention areadministered in conjunction with other agents, e.g. as hereinabovespecified, dosages of the co-administered agent will of course varydepending on the type of co-drug employed, on the specific drugemployed, on the condition being treated and so forth.

In accordance with the foregoing the present invention provides in a yetfurther aspect methods as defined above comprising co-administration,e.g. concomitantly or in sequence in any order (i.e. at the same time orsequentially in any order at different points in time), of atherapeutically effective amount of soluble CTLA4 molecules of theinvention, e.g. CTLA4Ig and/or L104EA29YIg, in free form or inpharmaceutically acceptable salt form, and a second drug substance, saidsecond drug substance being an immunosuppressant, immunomodulatory oranti-inflammatory drug, e.g. as indicated above.

Further provided are therapeutic combinations, e.g. a kit, comprising asoluble CTLA4 molecule, in free form or in pharmaceutically acceptablesalt form, to be used concomitantly or in sequence in any order (i.e. atthe same time or sequentially in any order at different points in time),with at least one pharmaceutical composition comprising anothertherapeutic agent. The kit may comprise instructions for itsadministration. The kits of the invention can be used in any method ofthe present invention.

The invention also provides methods for producing the soluble CTLA4mutant molecules of the invention. Expression of soluble CTLA4 mutantmolecules can be in prokaryotic cells or eukaryotic cells.

Prokaryotes most frequently are represented by various strains ofbacteria. The bacteria may be a gram positive or a gram negative.Typically, gram-negative bacteria such as E. coli are preferred. Othermicrobial strains may also be used. Sequences encoding soluble CTLA4mutant molecules can be inserted into a vector designed for expressingforeign sequences in prokaryotic cells such as E. coli. These vectorscan include commonly used prokaryotic control sequences which aredefined herein to include promoters for transcription initiation,optionally with an operator, along with ribosome binding site sequences,including such commonly used promoters as the beta-lactamase(penicillinase) and lactose (lac) promoter systems (Chang, et al.,(1977) Nature 198:1056), the tryptophan (trp) promoter system (Goeddel,et al., (1980) Nucleic Acids Res. 8:4057) and the lambda derived P_(L)promoter and N-gene ribosome binding site (Shimatake, et al., (1981)Nature 292:128).

Such expression vectors will also include origins of replication andselectable markers, such as a beta-lactamase or neomycinphosphotransferase gene conferring resistance to antibiotics, so thatthe vectors can replicate in bacteria and cells carrying the plasmidscan be selected for when grown in the presence of antibiotics, such asampicillin or kanamycin.

The expression plasmid can be introduced into prokaryotic cells via avariety of standard methods, including but not limited to CaCl₂-shock(Cohen, (1972) Proc. Natl. Acad. Sci. USA 69:2110, and Sambrook et al.(eds.), “Molecular Cloning: A Laboratory Manual”, 2nd Edition, ColdSpring Harbor Press, (1989)) and electroporation.

In accordance with the practice of the invention, eukaryotic cells arealso suitable host cells. Examples of eukaryotic cells include anyanimal cell, whether primary or immortalized, yeast (e.g., Saccharomycescerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), and plantcells. Myeloma, COS and CHO cells are examples of animal cells that maybe used as hosts. Particular CHO cells include, but are not limited to,DG44 (Chasin, et la., 1986 Som. Cell Molec. Genet. 12:555-556; Kolkekar1997 Biochemistry 36:10901-10909), CHO-K1 (ATCC No. CCL-61), CHO-K1Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAMR,Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B(GEIMG, Genova, IT), CHO-K1/SF designated ECACC 93061607 (CAMR,Salisbury, Wiltshire, UK), and RR-CHOK1 designated ECACC 92052129 (CAMR,Salisbury, Wiltshire, UK). Exemplary plant cells include tobacco (wholeplants, cell culture, or callus), corn, soybean, and rice cells. Corn,soybean, and rice seeds are also acceptable.

Nucleic acid sequences encoding the CTLA4 mutant molecules can also beinserted into a vector designed for expressing foreign sequences in aneukaryotic host. The regulatory elements of the vector can varyaccording to the particular eukaryotic host.

Commonly used eukaryotic control sequences for use in expression vectorsinclude promoters and control sequences compatible with mammalian cellssuch as, for example, CMV promoter (CDM8 vector) and avian sarcoma virus(ASV) (πLN vector). Other commonly used promoters include the early andlate promoters from Simian Virus 40 (SV40) (Fiers, et al., (1973) Nature273:113), or other viral promoters such as those derived from polyoma,Adenovirus 2, and bovine papilloma virus. An inducible promoter, such ashMTII (Karin, et al., (1982) Nature 299:797-802) may also be used.

Vectors for expressing CTLA4 mutant molecules in eukaryotes may alsocarry sequences called enhancer regions. These are important inoptimizing gene expression and are found either upstream or downstreamof the promoter region.

Examples of expression vectors for eukaryotic host cells include, butare not limited to, vectors for mammalian host cells (e.g., BPV-1, pHyg,pRSV, pSV2, pTK2 (Maniatis); pIES (Clontech); pRc/CMV2, pRc/RSV, pSFV1(Life Technologies); pVPakc Vectors, pCMV vectors, pSG5 vectors(Stratagene)), retroviral vectors (e.g., pFB vectors (Stratagene)),pCDNA-3 (Invitrogen) or modified forms thereof, adenoviral vectors;Adeno-associated virus vectors, baculovirus vectors, yeast vectors(e.g., pESC vectors (Stratagene)).

Nucleic acid sequences encoding CTLA4 mutant molecules can integrateinto the genome of the eukaryotic host cell and replicate as the hostgenome replicates. Alternatively, the vector carrying CTLA4 mutantmolecules can contain origins of replication allowing forextrachromosomal replication.

For expressing the nucleic acid sequences in Saccharomyces cerevisiae,the origin of replication from the endogenous yeast plasmid, the 2μcircle can be used. (Broach, (1983) Meth. Enz. 101:307). Alternatively,sequences from the yeast genome capable of promoting autonomousreplication can be used (see, for example, Stinchcomb et al., (1979)Nature 282:39); Tschemper et al., (1980) Gene 10:157; and Clarke et al.,(1983) Meth. Enz. 101:300).

Transcriptional control sequences for yeast vectors include promotersfor the synthesis of glycolytic enzymes (Hess et al., (1968) J. Adv.Enzyme Reg. 7:149; Holland et al., (1978) Biochemistry 17:4900).Additional promoters known in the art include the CMV promoter providedin the CDM8 vector (Toyama and Okayama, (1990) FEBS 268:217-221); thepromoter for 3-phosphoglycerate kinase (Hitzeman et al., (1980) J. Biol.Chem. 255:2073), and those for other glycolytic enzymes.

Other promoters are inducible because they can be regulated byenvironmental stimuli or by the growth medium of the cells. Theseinducible promoters include those from the genes for heat shockproteins, alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,enzymes associated with nitrogen catabolism, and enzymes responsible formaltose and galactose utilization.

Regulatory sequences may also be placed at the 3′ end of the codingsequences. These sequences may act to stabilize messenger RNA. Suchterminators are found in the 3′ untranslated region following the codingsequences in several yeast-derived and mammalian genes.

Exemplary vectors for plants and plant cells include, but are notlimited to, Agrobacterium T_(i) plasmids, cauliflower mosaic virus(CaMV), and tomato golden mosaic virus (TGMV).

General aspects of mammalian cell host system transformations have beendescribed by Axel (U.S. Pat. No. 4,399,216 issued Aug. 16, 1983).Mammalian cells can be transformed by methods including but not limitedto, transfection in the presence of calcium phosphate, microinjection,electroporation, or via transduction with viral vectors.

Methods for introducing foreign DNA sequences into eukaryote genomes,including plant and yeast genomes include; (1) mechanical methods, suchas microinjection of DNA into single cells or protoplasts, vortexingcells with glass beads in the presence of DNA, or shooting DNA-coatedtungsten or gold spheres into cells or protoplasts; (2) introducing DNAby making cell membranes permeable to macromolecules throughpolyethylene glycol treatment or subjection to high voltage electricalpulses (electroporation); or (3) the use of liposomes (containing cDNA)which fuse to cell membranes.

Once the CTLA4 mutant molecules of the inventions are expressed, theycan be harvested by methods well known in the art such as cell lysis(e.g. sonication, lysozyme and/or detergents) and protein recoveryperformed using standard protein purification means, e.g., affinitychromatography or ion-exchange chromatography, to yield substantiallypure product (R. Scopes in: “Protein Purification, Principles andPractice”, Third Edition, Springer-Verlag (1994); Sambrook et al.(eds.), “Molecular Cloning: A Laboratory Manual”, 2nd Edition, ColdSpring Harbor Press, (1989)). Expression of CTLA4 mutant molecules canbe detected by methods known in the art. For example, the mutantmolecules can be detected by Coomassie staining SDS-PAGE gels andimmunoblotting using antibodies that bind CTLA4.

EXAMPLES

The following Examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Example 1

The following provides a description of the methods used to generate thenucleotide sequences encoding the CTLA4 molecules of the invention.

A CTLA4Ig encoding plasmid was first constructed, and shown to expressCTLA4Ig molecules as described in U.S. Pat. Nos. 5,434,131, 5,885,579and 5,851,795. Then single-site mutant molecules (e.g., L104EIg) weregenerated from the CTLA4Ig encoding sequence, expressed and tested forbinding kinetics for various B7 molecules. The L104EIg nucleotidesequence (as included in the sequence shown in FIG. 18) was used as atemplate to generate the double-site CTLA4 mutant sequences (as includedin the sequences shown in FIGS. 19-22) which were expressed as proteinsand tested for binding kinetics. The double-site CTLA4 mutant sequencesinclude: L104EA29YIg, L104EA29LIg, L104EA29TIg, and L104EA29WIg.Triple-site mutants were also generated.

CTLA4Ig Construction

A genetic construct encoding CTLA4Ig comprising the extracellular domainof CTLA4 and an IgCgamma1 domain was constructed as described in U.S.Pat. Nos. 5,434,131, 5,844,095 and 5,851,795, the contents of which areincorporated by reference herein. The extracellular domain of the CTLA4gene was cloned by PCR using synthetic oligonucleotides corresponding tothe published sequence (Dariavach et al., Eur. Journ. Immunol.18:1901-1905 (1988)).

Because a signal peptide for CTLA4 was not identified in the CTLA4 gene,the N-terminus of the predicted sequence of CTLA4 was fused to thesignal peptide of oncostatin M (Malik et al., Mol. and Cell. Biol.9:2847 (1989)) in two steps using overlapping oligonucleotides. For thefirst step, the oligonucleotide,CTCAGTCTGGTCCTTGCACTCCTGTTTCCAAGCATGGCGAGCATGGCAATG CACGTGGCCCAGCC (SEQID NO:1) (which encoded the C terminal 15 amino acids from theoncostatin M signal peptide fused to the N terminal 7 amino acids ofCTLA4) was used as forward primer, and TTTGGGCTCCTGATCAGAATCTGGGCACGGTTG (SEQ ID NO: 2) (encoding amino acid residues 119-125 of theamino acid sequence encoding CTLA4 receptor and containing a Bcl Irestriction enzyme site) as reverse primer. The template for this stepwas cDNA synthesized from 1 micro g of total RNA from H38 cells (an HTLVII infected T-cell leukemic cell line provided by Drs. Salahudin andGallo, NCI, Bethesda, Md.). A portion of the PCR product from the firststep was reamplified, using an overlapping forward primer, encoding theN terminal portion of the oncostatin M signal peptide and containing aHind III restriction endonuclease site, CTAGCCACTGAAGCTTCACCAATGGGTGTACTGCTCACACA-GAGGACGCTGCTCAGTCTGGTCCTTGCACTC (SEQ ID NO: 3) and thesame reverse primer. The product of the PCR reaction was digested withHind III and Bcl I and ligated together with a Bcl 1/Xba I cleaved cDNAfragment encoding the amino acid sequences corresponding to the hinge,CH2 and CH3 regions of IgC(gamma)1 into the Hind III/Xba I cleavedexpression vector, CDM8 or Hind III/Xba I cleaved expression vector piLN(also known as πLN).

DNA encoding the amino acid sequence corresponding to CTLA4Ig has beendeposited with the ATCC under the Budapest Treaty on May 31, 1991, andhas been accorded ATCC accession number 68629.

CTLA4Ig Codon Based Mutagenesis

A mutagenesis and screening strategy was developed to identify mutantCTLA4Ig molecules that had slower rates of dissociation (“off” rates)from CD80 and/or CD86 molecules i.e. improved binding ability. In thisembodiment, mutations were carried out in and/or about the residues inthe CDR-1, CDR-2 (also known as the C′ strand) and/or CDR-3 regions ofthe extracellular domain of CTLA4 (as described in U.S. Pat. Nos.6,090,914, 5,773,253 and 5,844,095; in copending U.S. Patent ApplicationSer. No. 60/214,065; and by Peach, R. J., et al J Exp Med 1994180:2049-2058. A CDR-like region encompasses the each CDR region andextends, by several amino acids, upstream and/or downstream of the CDRmotif). These sites were chosen based on studies of chimeric CD28/CTLA4fusion proteins (Peach et al., J. Exp. Med., 1994, 180:2049-2058), andon a model predicting which amino acid residue side chains would besolvent exposed, and a lack of amino acid residue identity or homologyat certain positions between CD28 and CTLA4. Also, any residue which isspatially in close proximity (5 to 20 Angstrom Units) to the identifiedresidues is considered part of the present invention.

To synthesize and screen soluble CTLA4 mutant molecules with alteredaffinities for a B7 molecule (e.g. CD80, CD86), a two-step strategy wasadopted. The experiments entailed first generating a library ofmutations at a specific codon of an extracellular portion of CTLA4 andthen screening these by BIAcore analysis to identify mutants withaltered reactivity to B7. The Biacore assay system (Pharmacia,Piscataway, N.J.) uses a surface plasmon resonance detector system thatessentially involves covalent binding of either CD80Ig or CD86Ig to adextran-coated sensor chip which is located in a detector. The testmolecule can then be injected into the chamber containing the sensorchip and the amount of complementary protein that binds can be assessedbased on the change in molecular mass which is physically associatedwith the dextran-coated side of the sensor chip; the change in molecularmass can be measured by the detector system.

Specifically, single-site mutant nucleotide sequences were generatedusing nan-mutated (e.g., wild-type) DNA encoding CTLA4Ig (U.S. Pat. Nos.5,434,131, 5,844,095; 5,851,795; and 5,885,796; ATCC Accession No.68629) as a template. Mutagenic oligonucleotide PCR primer, weredesigned for random mutagenesis of a specific codon by allowing any baseat positions 1 and 2 of the codon, but only guanine or thymine atposition 3 (XXG/T or also noted as NNG/T), In this manner, a specificcodon encoding an amino acid could be randomly mutated to code for eachof the 20 amino acids. In that regard, XXG/T mutagenesis yields 32potential codona encoding each of the 20 amino acids. PCR productsencoding mutations in close proximity to the CDR3-like loop of CTLA4Ig(MYPPPY) (SEQ. ID NO:20), were digested with Sacl/Xbal and subclonedinto similarly cut CTLA4Ig (as included in FIG. 24) πLN expressionvector. This method was used to generate the single-site CTLA4 mutantmolecule L104EIg (as included in FIG. 18).

For mutagenesis in proximity to the CDR-1-like loop of CTLA4Ig, a silentNheI restriction site was first introduced 5′ to this loop, by PCRprimer-directed mutagenesis. PCR products were digested with NheI/XbaIand subcloned into similarly cut CTLA4Ig or L104EIg expression vectors.This method was used to generate the double-site CTLA4 mutant moleculeL104EA29YIg (as included in FIG. 19). In particular, the nucleic acidmolecule encoding the single-site CTLA4 mutant molecule, L104EIg, wasused as a template to generate the double-site CTLA4 mutant molecule,L104EA29YIg.

The double-site mutant nucleotide sequences encoding CTLA4 mutantmolecules, such as L104EA29YIg (deposited on Jun. 19, 2000 with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209 and accorded ATCC accession number PTA-2104),were generated by repeating the mutagenesis procedure described aboveusing L104EIg as a template. This method was used to generate numerousdouble-site mutants nucleotide sequences such as those encoding CTLA4molecules L104EA29YIg (as included in the sequence shown in FIG. 19),L104EA29LIg (as included in the sequence shown in FIG. 20), L104EA29TIg(as included in the sequence shown in FIG. 21), and L104EA29WIg (asincluded in the sequence shown in FIG. 22). Triple-site mutants, such asthose encoding L104EA29YS25KIg, L104EA29YS25NIg and L104EA29YS25RIg,were also generated.

The soluble CTLA4 molecules were expressed from the nucleotide sequencesand used in the phase II clinical studies described in Example 3, infra.

As those skilled-in-the-art will appreciate, replication of nucleic acidsequences, especially by PCR amplification, easily introduces basechanges into DNA strands. However, nucleotide changes do not necessarilytranslate into amino acid changes as some codons redundantly encode thesame amino acid. Any changes of nucleotide from the original or wildtypesequence, silent (i.e. causing no change in the translated amino acid)or otherwise, while not explicitly described herein, are encompassedwithin the scope of the invention.

Example 2

The following Example provides a description of the screening methodsused to identify the single- and double-site mutant CTLA polypeptides,expressed from the constructs described in Example 1, that exhibited ahigher binding avidity for B7 molecules, compared to non-mutated CTLA4Igmolecules.

Current in vitro and in vivo studies indicate that CTLA4Ig by itself isunable to completely block the priming of antigen specific activated Tcells. In vitro studies with CTLA4Ig and either monoclonal antibodyspecific for CD80 or CD86 measuring inhibition of T cell proliferationindicate that anti-CD80 monoclonal antibody did not augment CTLA4Iginhibition. However, anti-CD86 monoclonal antibody did augment theinhibition, indicating that CTLA4Ig was not as effective at blockingCD86 interactions. These data support earlier findings by Linsley et al.(Immunity, (1994), 1:793-801) showing inhibition of CD80-mediatedcellular responses required approximately 100 fold lower CTLA4Igconcentrations than for CD86-mediated responses. Based on thesefindings, it was surmised that soluble CTLA4 mutant molecules having ahigher avidity for CD86 than wild type CTLA4 should be better able toblock the priming of antigen specific activated cells than CTLA4Ig.

To this end, the soluble CTLA4 mutant molecules described in Example 1above were screened using a novel screening procedure to identifyseveral mutations in the extracellular domain of CTLA4 that improvebinding avidity for CD80 and CD86. This screening strategy provided aneffective method to directly identify mutants with apparently slower“off” rates without the need for protein purification or quantitationsince “off” rate determination is concentration independent (O'Shannessyet al., (1993) Anal. Biochem., 212:457-468).

COS cells were transfected with individual miniprep purified plasmid DNAand propagated for several days. Three day conditioned culture media wasapplied to BIAcore biosensor chips (Pharmacia Biotech AB, Uppsala,Sweden) coated with soluble CD80Ig or CD86Ig. The specific binding anddissociation of mutant proteins was measured by surface plasmonresonance (O'Shannessy, D. J., et al., 1997 Anal. Biochem. 212:457-468).All experiments were run on BIAcore™ or BIAcore™ 2000 biosensors at 25°C. Ligands were immobilized on research grade NCM5 sensor chips(Pharmacia) using standard N-ethyl-N′-(dimethylaminopropyl)carbodiimidN-hydroxysuccinimide coupling (Johnsson, B., et al. (1991)Anal. Biochem. 198: 268-277; Khilko, S. N., et al. (1993) J. Biol. Chem268:5425-15434).

Screening Method

COS cells grown in 24 well tissue culture plates were transientlytransfected with mutant CTLA4Ig. Culture media containing secretedsoluble mutant CTLA4Ig was collected 3 days later.

Conditioned COS cell culture media was allowed to flow over BIAcorebiosensor chips derivitized with CD86Ig or CD80Ig (as described inGreene et al., 1996 J. Biol. Chem. 271:26762-26771), and mutantmolecules were identified with off-rates slower than that observed forwild type CTLA4Ig. The DNAs corresponding to selected media samples weresequenced and more DNA prepared to perform larger scale COS celltransient transfection, from which CTLA4Ig mutant protein was preparedfollowing protein A purification of culture media.

BIAcore analysis conditions and equilibrium binding data analysis wereperformed as described in J. Greene et al. 1996 J. Biol. Chem.271:26762-26771 and in U.S. patent application Ser. Nos. 09/579,927, and60/214,065 which are herein incorporated by reference.

BIAcore Data Analysis

Senosorgram baselines were normalized to zero response units (RU) priorto analysis. Samples were run over mock-derivatized flow cells todetermine background RU values due to bulk refractive index differencesbetween solutions. Equilibrium dissociation constants (K_(d)) werecalculated from plots of R_(eq) versus C, where R_(eq) is thesteady-state response minus the response on a mock-derivatized chip, andC is the molar concentration of analyte. Binding curves were analyzedusing commercial nonlinear curve-fitting software (Prism, GraphPADSoftware).

Experimental data were first fit to a model for a single ligand bindingto a single receptor (1-site model, i.e., a simple langmuir system,A+B⇄AB), and equilibrium association constants (K_(d)=[A]·[B]\[AB]) werecalculated from the equation R=R_(max)·C/(K_(d)+C). Subsequently, datawere fit to the simplest two-site model of ligand binding (i.e., to areceptor having two non-interacting independent binding sites asdescribed by the equation R=R_(max1)·C\(K_(d1)+C)+R_(max2)·C\(K_(d2)+C).

The goodness-of-fits of these two models were analyzed visually bycomparison with experimental data and statistically by an F test of thesums-of-squares. The simpler one-site model was chosen as the best fit,unless the two-site model fit significantly better (p<0.1).

Association and disassociation analyses were performed using BIAevaluation 2.1 Software (Pharmacia). Association rate constants k_(on)were calculated in two ways, assuming both homogenous single-siteinteractions and parallel two-site interactions. For single-siteinteractions, k_(on) values were calculated according to the equationR_(t)=R_(eq)(1−exp^(−ks(t−t) ₀), where R_(t) is a response at a giventime, t; R_(eq) is the steady-state response; t₀ is the time at thestart of the injection; and k_(s)=dR/dt=k_(on)·Ck_(off), where C is aconcentration of analyte, calculated in terms of monomeric bindingsites. For two-site interactions k_(on) values were calculated accordingto the equation R_(t)=R_(eq1)(1−exp^(−ks1(t−t)₀)+R_(eq2)(1−exp^(−ks2(t−t) ₀). For each model, the values of k_(on)were determined from the calculated slope (to about 70% maximalassociation) of plots of k_(s) versus C.

Dissociation data were analyzed according to one site (AB=A+B) or twosite (AiBj=Ai⁺ Bj) models, and rate constants (k_(off)) were calculatedfrom best fit curves. The binding site model was used except when theresiduals were greater than machine background (2-10RU, according tomachine), in which case the two-binding site model was employed.Half-times of receptor occupancy were calculated using the relationshipt_(1/2)=0.693/k_(off).

Flow Cytometry

Murine mAb L307.4 (anti-CD80) was purchased from Becton Dickinson (SanJose, Calif.) and IT2.2 (anti-B7-0 [also known as CD86]), fromPharmingen (San Diego, Calif.). For immunostaining, CD80-positive and/orCD86-positive CHO cells were removed from their culture vessels byincubation in phosphate-buffered saline (PBS) containing 10 mM EDTA. CHOcells (1-10×10⁵) were first incubated with mAbs or immunoglobulin fusionproteins in DMEM containing 10% fetal bovine serum (FBS), then washedand incubated with fluorescein isothiocyanate-conjugated goat anti-mouseor anti-human immunoglobulin second step reagents (Tago, Burlingame,Calif.). Cells were given a final wash and analyzed on a FACScan (BectonDickinson).

SDS-PAGE and Size Exclusion Chromatography

SDS-PAGE was performed on Tris/glycine 4-20% acrylamide gels (Novex, SanDiego, Calif.). Analytical gels were stained with Coomassie Blue, andimages of wet gels were obtained by digital scanning. CTLA4Ig (25 μg)and L104EA29YIg (25 μg) were analyzed by size exclusion chromatographyusing a TSK-GEL G300 SW_(XL) column (7.8×300 mm, Tosohaas,Montgomeryville, Pa.) equilibrated in phosphate buffered salinecontaining 0.02% NAN₃ at a flow rate of 1.0 ml/min.

CTLA4X_(C120S) and L104EA29YX_(C120S)

Single chain CTLA4X_(C120S) was prepared as previously described(Linsley et al., (1995) J. Biol. Chem., 270:15417-15424). Briefly, anoncostatin M CTLA4 (OMCTLA4) expression plasmid was used as a template,the forward primer, GAGGTGATAAAGCTTCACCAATGGGTGTACTGCTCACACAG (SEQ IDNO: 4) was chosen to match sequences in the vector; and the reverseprimer, GTGGTGTATTGGTCTAGATCAATCAGAATCTGGGCACGGTTC (SEQ ID NO: 5)corresponded to the last seven amino acids (i.e. amino acids 118-124) inthe extracellular domain of CTLA4, and contained a restriction enzymesite, and a stop codon (TGA). The reverse primer specified a C120S(cysteine to serine at position 120) mutation. In particular, thenucleotide sequence GCA (nucleotides 34-36) of the reverse primer shownabove is replaced with one of the following nucleotide sequences: AGA,GGA, TGA, CGA, ACT, or GCT. As persons skilled in the art willunderstand, the nucleotide sequence GCA is a reversed complementarysequence of the codon TGC for cysteine. Similarly, the nucleotidesequences AGA, GGA, TGA, CGA, ACT, or GCT are the reversed complementarysequences of the codons for serine. Polymerase chain reaction productswere digested with HindIII/XbaI and directionally subcloned into theexpression vector RLN (Bristol-Myers Squibb Company, Princeton, N.J.).L104EA29YX_(Cl20S) was prepared in an identical manner. Each constructwas verified by DNA sequencing.

Identification and Biochemical Characterization of High Avidity Mutants

Twenty four amino acids were chosen for mutagenesis and the resulting˜2300 mutant proteins assayed for CD86Ig binding by surface plasmonresonance (SPR; as described, supra). The predominant effects ofmutagenesis at each site are summarized in Table II, infra. Randommutagenesis of some amino acids in the CDR-1 region (S25-R33) apparentlydid not alter ligand binding. Mutagenesis of E31 and R33 and residuesM97-Y102 apparently resulted in reduced ligand binding. Mutagenesis ofresidues, S25, A29, and T30, K93, L96, Y103, L104, and G105, resulted inproteins with slow “on” and/or slow “off” rates. These results confirmprevious findings that residues in the CDR-1 (S25-R33) region, andresidues in or near M97-Y102 influence ligand binding (Peach et al.,(1994) J. Exp. Med., 180:2049-2058).

Mutagenesis of sites S25, T30, K93, L96, Y103, and G105 resulted in theidentification of some mutant proteins that had slower “off” rates fromCD86Ig. However, in these instances, the slow “off” rate was compromisedby a slow “on” rate that resulted in mutant proteins with an overallavidity for CD86Ig that was apparently similar to that seen with wildtype CTLA4Ig. In addition, mutagenesis of K93 resulted in significantaggregation that may have been responsible for the kinetic changesobserved.

Random mutagenesis of L104 followed by COS cell transfection andscreening by SPR of culture media samples over immobilized CD86Igyielded six media samples containing mutant proteins with approximately2-fold slower “off” rates than wild type CTLA4Ig. When the correspondingcDNA of these mutants were sequenced, each was found to encode a leucineto glutamic acid mutation (L104E). Apparently, substitution of leucine104 to aspartic acid (L104D) did not affect CD86Ig binding.

Mutagenesis was then repeated at each site listed in Table II, this timeusing L104E as the PCR template instead of wild type CTLA4Ig, asdescribed above. SPR analysis, again using immobilized CD86Ig,identified six culture media samples from mutagenesis of alanine 29 withproteins having approximately 4-fold slower “off” rates than wild typeCTLA4Ig. The two slowest were tyrosine substitutions (L104EA29Y), twowere leucine (L104EA29L), one was tryptophan (L104EA29W), and one wasthreonine (L104EA29T). Apparently, no slow “off” rate mutants wereidentified when alanine 29 was randomly mutated, alone, in wild typeCTLA4Ig.

The relative molecular mass and state of aggregation of purified L104Eand L104EA29YIg was assessed by SDS-PAGE and size exclusionchromatography. L104EA29YIg (˜1 μg; lane 3) and L104EIg (˜1 μg; lane 2)apparently had the same electrophoretic mobility as CTLA4Ig (˜1 μg;lane 1) under reducing (50 kDa; +βME; plus 2-mercaptoethanol) andnon-reducing (˜100 kDa; ˜βME) conditions (FIG. 25A). Size exclusionchromatography demonstrated that L104EA29YIg (FIG. 25C) apparently hadthe same mobility as dimeric CTLA4Ig (FIG. 25B). The major peaksrepresent protein dimer while the faster eluting minor peak in FIG. 25Brepresents higher molecular weight aggregates. Approximately 5.0% ofCTLA4Ig was present as higher molecular weight aggregates but there wasno evidence of aggregation of L104EA29YIg or L104EIg. Therefore, thestronger binding to CD86Ig seen with L104EIg and LI04EA29YIg could notbe attributed to aggregation induced by mutagenesis.

Equilibrium and Kinetic Binding Analysis

Equilibrium and kinetic binding analysis was performed on protein Apurified CTLA4Ig, L104EIg, and L104EA29YIg using surface plasmonresonance (SPR). The results are shown in Table I, infra. Observedequilibrium dissociation constants (K_(d); Table I) were calculated frombinding curves generated over a range of concentrations (5.0-200 nM).L104EA29YIg binds more strongly to CD86Ig than does L104EIg or CTLA4Ig.The lower K_(d) of L104EA29YIg (3.21 nM) than L104EIg (6.06 nM) orCTLA4Ig (13.9 nM) indicates higher binding avidity of L104EA29YIg toCD86Ig. The lower K_(d) of L104EA29YIg (3.66 nM) than L104EIg (4.47 nM)or CTLA4Ig (6.51 nM) indicates higher binding avidity of L104EA29YIg toCD80Ig.

Kinetic binding analysis revealed that the comparative “on” rates forCTLA4Ig, L104EIg, and L104EA29YIg binding to CD80 were similar, as werethe “on” rates for CD86Ig (Table I). However, “off” rates for thesemolecules were not equivalent (Table I). Compared to CTLA4Ig,L104EA29YIg had approximately 2-fold slower “off” rate from CD80Ig, andapproximately 4-fold slower “off” rate from CD86Ig. L104E had “off”rates intermediate between L104EA29YIg and CTLA4Ig. Since theintroduction of these mutations did not significantly affect “on” rates,the increase in avidity for CD80Ig and CD86Ig observed with L104EA29YIgwas likely primarily due to a decrease in “off” rates.

To determine whether the increase in avidity of L104EA29YIg for CD86Igand CD80Ig was due to the mutations affecting the way each monomerassociated as a dimer, or whether there were avidity enhancingstructural changes introduced into each monomer, single chain constructsof CTLA4 and L104EA29Y extracellular domains were prepared followingmutagenesis of cysteine 120 to serine as described supra, and by Linsleyet al., (1995) J. Biol. Chem., 270:15417-15424 (84). The purifiedproteins CTLA4X_(C120S) and L104EA29YX_(C120S) were shown to bemonomeric by gel permeation chromatography (Linsley et al., (1995),supra), before their ligand binding properties were analyzed by SPR.Results showed that binding affinity of both monomeric proteins forCD86Ig was approximately 35-80-fold less than that seen for theirrespective dimers (Table I). This supports previously published dataestablishing that dimerization of CTLA4 was required for high avidityligand binding (Greene et al., (1996) J. Biol. Chem., 271:26762-26771).

L104EA29YX_(C120S) bound with approximately 2-fold higher affinity thanCTLA4X_(C120S) to both CD80Ig and CD86Ig. The increased affinity was dueto approximately 3-fold slower rate of dissociation from both ligands.Therefore, stronger ligand binding by L104EA29Y was most likely due toavidity enhancing structural changes that had been introduced into eachmonomeric chain rather than alterations in which the molecule dimerized.

Location and Structural Analysis of Avidity Enhancing Mutations

The solution structure of the extracellular IgV-like domain of CTLA4 hasrecently been determined by NMR spectroscopy (Metzler et al., (1997)Nature Struct. Biol., 4:527-531). This allowed accurate location ofleucine 104 and alanine 29 in the three dimensional fold (FIG. 26 leftand right depictions). Leucine 104 is situated near the highly conservedMYPPPY (SEQ. ID NO:20) amino acid sequence. Alanine 29 is situated nearthe C-terminal end of the CDR-1 (S25-R33) region, which is spatiallyadjacent to the MYPPPY (SEQ. ID NO:20) region. While there issignificant interaction between residues at the base of these tworegions, there is apparently no direct interaction between L104 and A29although they both comprise part of a contiguous hydrophobic core in theprotein. The structural consequences of the two avidity enhancingmutants were assessed by modeling. The A29Y mutation can be easilyaccommodated in the cleft between the CDR-1 (S25-R33) region and theMYPPPY (SEQ. ID NO:20) region, and may serve to stabilize theconformation of the MYPPPY (SEQ. ID NO:20) region. In wild type CTLA4,L104 forms extensive hydrophobic interactions with L96 and V94 near theMYPPPY (SEQ. ID NQ:20) region. It highly unlikely that the giutamic acidmutation adopts a conformation similar to that of L104 for two reasons.First, there is insufficient space to accommodate the longer glutamicacid side chain in the structure without significant perturbation to theCDR-1 (S25-R33 region). Second, the energetic costs of burying thenegative charge of the glutamic acid side chain in the hydrophobicregion would be large. Instead, modeling studies predict that thegiutemic acid side chain flips out on to the surface where its chargecan be stabiiized by solvation. Such a conformational change can easilybe accommodated by G105, with minimal distortion to other residues inthe regions.

Binding of High Avidity Mutants to CHO Cells Expressing CD80 or CD86

FACS analysis (FIG. 27) of CTLA4Ig and mutant molecules binding tostably transfected CD80+ and CD86+CHO cells was performed as describedherein. CD80-positive and CD86-positive CHO cells were incubated withincreasing concentrations of CTLA4Ig, L104EA29YIg, or L104EIg, and thenwashed. Bound immunoglobulin fusion protein was detected usingfluorescein isothiocyanate-conjugated goat anti-human immunoglobulin.

As shown in FIG. 27, CD80-positive or CD86-positive CHO cells (1.5×10⁵)were incubated with the indicated concentrations of CTLA4Ig (closedsquares), L104EA29YIg (circles), or L104EIg (triangles) for 2 hr. at 23°C., washed, and incubated with fluorescein isothiocyanate-conjugatedgoat anti-human immunoglobulin antibody. Binding on a total of 5,000viable cells was analyzed (single determination) on a FACScan, and meanfluorescence intensity (MFI) was determined from data histograms usingPC-LYSYS. Data were corrected for background fluorescence measured oncells incubated with second step reagent only (MFI=7). Control L6 mAb(80 μg/ml) gave MFI<30. These results are representative of fourindependent experiments.

Binding of L104EA29YIg, L104EIg, and CTLA4Ig to human CD80-transfectedCHO cells is approximately equivalent (FIG. 27A). L104EA29YIg andL104EIg bind more strongly to CHO cells stably transfected with humanCD86 than does CTLA4Ig (FIG. 27B).

Functional Assays

Human CD4-positive T cells were isolated by immunomagnetic negativeselection (Linsley et al., (1992) J. Exp. Med. 176:1595-1604). IsolatedCD4-positive T cells were stimulated with phorbal myristate acetate(PMA) plus CD80-positive or CD86-positive CHO cells in the presence oftitrating concentrations of inhibitor. CD4-positive T cells(8-10×10⁴/well) were cultured in the presence of 1 nM PMA with orwithout irradiated CHO cell stimulators. Proliferative responses weremeasured by the addition of 1 μCi/well of [3H]thymidine during the final7 hours of a 72 hour culture. Inhibition of PMA plus CD80-positive CHO,or CD86-positive CHO, stimulated T cells by L104EA29YIg and CTLA4Ig wasperformed. The results are shown in FIG. 28. L104EA29YIg inhibitsproliferation of CD80-positive PMA treated CHO cells more than CTLA4Ig(FIG. 28A). L104EA29YIg is also more effective than CTLA4Ig atinhibiting proliferation of CD86-positive PMA treated CHO cells (FIG.28B). Therefore, L104EA29YIg is a more potent inhibitor of both CD80-and CD86-mediated costimulation of T cells.

FIG. 29 shows inhibition by L104EA29YIg and CTLA4Ig of allostimulatedhuman T cells prepared above, and further allostimulated with a human Blymphoblastoid cell line (LCL) called PM that expressed CD80 and CD86 (Tcells at 3.0×10⁴/well and PM at 8.0×10³/well). Primary allostimulationoccurred for 6 days, then the cells were pulsed with ³H-thymidine for 7hours, before incorporation of radiolabel was determined.

Secondary allostimulation was performed as follows. Seven day primaryallostimulated T cells were harvested over lymphocyte separation medium(LSM) (ICN, Aurora, Ohio) and rested for 24 hours. T cells were thenrestimulated (secondary), in the presence of titrating amounts ofCTLA4Ig or L104EA29YIg, by adding PM in the same ratio as above.Stimulation occurred for 3 days, then the cells were pulsed withradiolabel and harvested as above. The effect of L104EA29YIg on primaryallostimulated T cells is shown in FIG. 29A. The effect of L104EA29YIgon secondary allostimulated T cells is shown in FIG. 29B. L104EA29YIginhibits both primary and secondary T cell proliferative responsesbetter than CTLA4Ig.

To measure cytokine production (FIG. 30), duplicate secondaryallostimulation plates were set up. After 3 days, culture media wasassayed using ELISA kits (Biosource, Camarillo, Calif.) using conditionsrecommended by the manufacturer. L104EA29YIg was found to be more potentthan CTLA4Ig at blocking T cell IL-2, IL-4, and Y-IFN (gamma-IFN)cytokine production following a secondary allogeneic stimulus (FIGS.30A-C). The effects of L104EA29YIg and CTLA4Ig on monkey mixedlymphocyte response (MLR) are shown in FIG. 31. Peripheral bloodmononuclear cells (PBMC'S; 3.5×10⁴ cells/well from each monkey) from 2monkeys were purified over lymphocyte separation medium (LSM) and mixedwith 2 μg/ml phytohemaglutinin (PHA). The cells were stimulated 3 daysthen pulsed with radiolabel 16 hours before harvesting. L104EA29YIginhibited monkey T cell proliferation better than CTLA4Ig.

TABLE I Equilibrium and apparent kinetic constants are given in thefollowing table (values are means ± standard deviation from threedifferent experiments) Immo- bilized k_(on) (×10⁵) k_(off) (×10⁻³)Protein Analyte M⁻¹ S⁻¹ S⁻¹ K_(d) nM CD80Ig CTLA4Ig 3.44 ± 0.29 2.21 ±0.18 6.51 ± 1.08 CD80Ig L104EIg 3.02 ± 0.05 1.35 ± 0.08 4.47 ± 0.36CD80Ig L104EA29YIg 2.96 ± 0.20 1.08 ± 0.05 3.66 ± 0.41 CD80IgCTLA4X_(C120S) 12.0 ± 1.0  230 ± 10  195 ± 25  CD80Ig L104EA29YX_(C120S) 8.3 ± 0.26 71 ± 5  85.0 ± 2.5  CD86Ig CTLA4Ig 5.95 ± 0.57 8.16 ± 0.5213.9 ± 2.27 CD86Ig L104EIg 7.03 ± 0.22 4.26 ± 0.11 6.06 ± 0.05 CD86IgL104EA29YIg 6.42 ± 0.40 2.06 ± 0.03 3.21 ± 0.23 CD86Ig CTLA4X_(C120S)16.5 ± 0.5  840 ± 55  511 ± 17  CD86Ig L104EA29YX_(C120S) 11.4 ± 1.6 300 ± 10  267 ± 29 

TABLE II The effect on CD86Ig binding by mutagenesis of CTLA4Ig at thesites listed was determined by SPR, described supra. The predominanteffect is indicated with a “+” sign. Effects of Mutagenesis MutagenesisNo Apparent Slow “on” rate/ Reduced ligand Site Effect slow “off ratebinding S25 + P26 + G27 + K28 + A29 + T30 + E31 + R33 + K93 + L96 +M97 + Y98 + P99 + P100 + P101 + Y102 + Y103 + L104 + G105 + I106 +G107 + Q111 + Y113 + I115 +

Example 3

The following provides a description of phase II clinical studies ofhuman patients administered soluble CTLA4 mutant molecule L104EA29YIg(also known as L104EA29YIG or LEA) or CTLA4Ig, to relieve at least onesymptom associated with rheumatoid arthritis, including reducing: jointswelling, joint tenderness, inflammation, morning stiffness, and pain.The CTLA4Ig molecule used herein begins with methionine at position +1(or alternatively with alanine at position −1) and ends with lysine atposition +357 as shown in FIG. 24. DNA encoding an embodiment of theCTLA4Ig molecule has been deposited as ATCC 68629. The L104EA29YIgmolecule used herein begins with methionine at position +1 (oralternatively with alanine at position −1) and ends with lysine atposition +357 as shown in FIG. 19. DNA encoding an embodiment of theL104EA29YIg molecule has been deposited as ATCC PTA 2104.

Additionally, the following provides a description of human patientsadministered L104EA29YIg or CTLA4Ig to relieve at least one biologicalsurrogate marker associated with rheumatoid arthritis, includingreducing erythrocyte sedimentation rates, and serum levels of C-reactiveprotein and/or IL2 receptor.

Patient Cohorts

A total of 214 patients, including 54 males and 160 females,participated in the study (FIGS. 1A, 1B). The patients at baseline had amean disease duration of 3.4 (+2.0) years and had failed at least oneDisease Modifying Antirheumatic Drug (DMARD). Stable NonsteroidalAnti-inflammatory Drugs (NSAIDS) or steroids (≦10 mg/day) were permittedand concomitant DMARDS were prohibited. The patients were randomizedinto groups of 25 to 32 patients per treatment group. Thirty-twopatients received a placebo, 92 received L104EA29YIg, and 90 receivedCTLA4Ig. The patients who followed protocol guidelines and did notdiscontinue before day 57 received a total of 4 intravenous infusions,one infusion each on days 1, 15, 29, and 57. All patients were evaluatedon days 1, 15, 29, 43, 57, 71, and 85. The doses administered included0.5, 2.0, or 10.0 mg/kg of L104EA29YIg (denoted as LEA.5, LEA2 and LEA10, respectively in FIGS. 1A-1E) or of CTLA4Ig (denoted as CTLA.5, CTLA2and CTLA10, respectively in FIGS. 1A-1E).

All subjects were monitored for peri-infusional adverse events andglobal safety by answering a questionnaire listing potential adverseevents. The patients were questioned about potential adverse events thatmay have occurred within twenty-four hours post-infusion. In addition,the patients were encouraged to spontaneously report any adverse eventsthat they experienced. The physicians routinely monitored laboratorysamples from the patients for abnormalities in blood chemistry andhematology e.g. assessed the levels of inflammatory response mediatorssuch as cytokines (TNF, IL-6), tryptase and complement. The primaryendpoint was the proportion of subjects meeting the ACR 20 criteria onday 85.

Storage of Test Material

The CTLA4Ig and L104EA29YIg were supplied in single-use glass vialscontaining 200 mg/vial of CTLA4Ig or 100 mg/vial of L104EA29YIg,respectively. Prior to infusion, the CTLA4Ig and L104EA29YIg werediluted to a final concentration of 25 mg/ml with sterile water forinjection (SWFI).

Administration Protocol

All infusions were administered intravenously over 1 hour (FIGS. 1through 17). All subjects received at least one infusion of studymedication.

-   Group 1: 32 patients, CTLA4Ig or L104EA29YIg matching placebo.-   Group 2: 26 patients; dosage 0.5 mg/kg of CTLA4Ig.-   Group 3: 32 patients; dosage 2.0 mg/kg of CTLA4Ig.-   Group 4: 32 patients; dosage 10.0 mg/kg of CTLA4Ig.-   Group 5: 32 patients; dosage 0.5 mg/kg of L104EA29YIg.-   Group 6: 29 patients; dosage 2.0 mg/kg of L104EA29YIg.-   Group 7: 31 patients; dosage 10.0 mg/kg of L104EA29YIg.    Clinical Monitoring

Patients were evaluated for baseline symptoms of disease activity priorto receiving any infusions. These baseline evaluations included: jointswelling, joint tenderness, inflammation, morning stiffness, diseaseactivity evaluated by patient and physician as well as disabilityevaluated by Health Questionnaire Assessment (HAQ) (reported as aphysical function score in FIG. 1C), and pain (FIGS. 1A to 1D).Additionally, the baseline evaluations included erythrocytesedimentation rates (ESR), and serum levels of C-reactive protein (CRP)and soluble IL-2 receptor (IL-2r) (FIGS. 1C and 1D).

The clinical response studies were based on the criteria established bythe American College of Rheumatology (ACR). A subject satisfied theACR20 criterion if there was a 20 percent improvement in tender andswollen joint counts and 20 percent improvement in three of the fiveremaining symptoms measured, such as patient and physician globaldisease changes, pain, disability, and an acute phase reactant (Felson,D. T., et al., 1993 Arthritis and Rheumatism 36:729-740; Felson, D. T.,et al., 1995 Arthritis and Rheumatism 38:1-9). Similarly, a subjectsatisfied the ACR50 or ACR70 criterion if there was a 50 or 70 percentimprovement, respectively, in tender and swollen joint counts and 50 or70 percent improvement, respectively, in three of the five remainingsymptoms measured, such as patient and physician global disease changes,pain, physical disability, and an acute phase reactant such as CRP orESR.

Biomarkers

Potential biomarkers of disease activity (rheumatoid factor, CRP, ESR,soluble IL-2R, soluble ICAM-1, soluble E-selectin, and MMP-3) were alsoassessed. Validated enzyme immunoassay (EIA) methods were used todetermine the serum concentration of IL-2sRα, sICAM-1, sE-selectin andMMP-3. TNFα and IL-6 were assessed at infusion pre and 2 hours post, ifnecessary.

IL-2sRα, sICAM-1, and sE-selectin were measured using commerciallyavailable colorimetric EIA kits from R&D Systems, Inc. (Minneapolis,Minn.). The lower and upper limits of quantitation were 312-20,000μg/mL, 40-907 ng/mL and 10-206 ng/mL, respectively. The inter-assaycoefficient of variation ranged from 4.48-8.4%, 3.8-5.0% and 5.5-9.0%respectively. According to the kit manufacturer, normal serum valuesrange from 676-2,132 μg/mL, respectively.

MMP-3 was measured using a commercially available colorimetric EIA kitfrom Amersham Pharmacia Biotech (Piscataway, N.J.). The lower and upperlimits of quantitation were 30-7,680 ng/mL. The inter-assay coefficientof variation ranged from 6.3-10.6%. According to the kit manufacturer,normal serum values range from 28-99 ng/mL.

IL-6 and TNFα were measured using commercially availablechemiluminescent EIA kits from R&D Systems, Inc. (Minneapolis, Minn.).The lower and upper limits of quantitation were 0.3-3,000 pg/mL and0.7-7,000 pg/mL, respectively. The inter-assay coefficient of variationranged from 3.1-5.7% and 6.4-20.7%, respectively. According to the kitmanufacturer, normal serum values range from <0.3-12 pg/mL and <0.7-7.5pg/mL.

Antibody Testing

Serum samples were obtained for assessment of drug-specific antibodiesprior to dosing on day 1, and approximately on days 15, 29, 57, 85 and169. Due to high, preexisting titers directed to the immunoglobulin (Ig)portion of the molecule, specific antibody formation against CTLA4Ig andL104EA29YIG without Ig constant regions was also assessed.

Ninety-six well Immulon II ELISA plates (Dynex, Chantilly, Va.) werecoated with CTLA4Ig, CTLA4Ig without the Ig constant regions,L104EA29YIG, or L104EA29YIG without the Ig constant regions at 2, 4, 2,or 1 μg/ml in phosphate buffered saline (PBS), respectively, andincubated overnight at 2-8° C. The plates were washed with PBScontaining 0.05% Tween 20 and blocked for 1 hour at 37° C. with PBScontaining 1% bovine serum albumin (BSA). The plates were then washedand serial dilutions of the test sera or quality control (QC) sera wereadded to the appropriate wells and incubated for 2 hours at 37° C. Serawas diluted threefold in PBS with 0.25% BSA and 0.05% Tween 20 startingat a 1:10 dilution. Plates were washed and analkaline-phosphatase-conjugated goat anti-human kappa and lambda(Southern Biotechnology Associates, Inc., Birmingham, Ala.) antibodycocktail was added. Following a 1-hour incubation at 37° C., the plateswere washed and 1 mg/ml para-nitrophenyl phosphate in diethanolaminebuffer was added to each well. After 30 minutes at 25° C., the reactionswere stopped with 3N NaOH and the absorbance (dual wavelength: 405 nmand 550 nm) was recorded. Results were expressed as endpoint titer(EPT), defined as the reciprocal of the highest dilution that resultedin an absorbance reading fivefold greater than or equal to the meanplate-background absorbance. Plate background was determined as theabsorbance measurement recorded in the absence of serum. Values wereconsidered positive for seroconversion if they were at least two serialdilutions (ninefold) or greater relative to predose EPT values. Serum QCsamples positive for either CTLA4Ig- or L104EA29YIG-specific antibodieswere generated from immunized monkeys. An aliquot of the appropriate QCsample was assayed during each analytical run. Analytical runs wereaccepted only when the QC samples were within the assay acceptancecriteria.

Results

CTLA4Ig and L104EA29YIg were generally well-tolerated at alldose-levels. Peri-infusional adverse events were similar across all dosegroups, with the exception of headaches. Headache response of patientson day 85 increased dose-dependently 23%, 44%, and 53% inCTLA4Ig-treated patients, and 34%, 45%, and 61% in L104EA29YIg-treatedpatients, at 0.5, 2.0, and 10.0 mg/kg respectively. In contrast, 31% ofthe patients administered placebos experienced headaches.

The percent of patients that discontinued from the clinical study due toarthritis flares and other adverse events is summarized in FIG. 2. Amuch higher percentage of patients on placebo discontinued treatment dueto arthritis flare. The CTLA4Ig treated patients discontinued treatmentless with increasing doses. Very few patients treated with L104EA29YIgdiscontinued treatment. These results indicate a good inversedose-dependent response for CTLA4Ig, and a stronger therapeutic responsewith L104EA29YIg therapy.

The ACR-20, -50, and -70 responses of patients treated with CTLA4Ig,L104EA29YIg, or placebo at day 85 are summarized in FIG. 3A. Similarly,FIGS. 3B and C describe the ACR-20 responses with 95% confidence limits.The responses appear to be dose-dependent with a clear significantresponse at 10 mg/kg per body weight of the patient.

The percent of patients having reduced swollen and tender joint countscompared to the patients having no response to treatment with CTLA4Ig,L104EA29YIg, or placebo, is shown in FIGS. 4A and B. The therapeuticresponses appear to be dose-dependent. A larger percentage of patientsshow improvement of 20, 50, 70, and even 100% in the 2 and 10 mg/kggroups for both products.

The percent of patients having reduced pain, disease activity evaluatedby patient and physician mean score units with CTLA4Ig, L104EA29YIg, orplacebo, is shown in FIGS. 5A, B, C, and D. The therapeutic responses,as monitored by the Likert scale, appear to be dose-dependent in favorof the active treatment groups as compared to placebo on day 85. TheLikert scale is a validated verbal rating scale using adjectives to rankthe symptoms (The American College of Rheumatology Preliminary Core Setof Disease Activity Measures for Rheumatoid Arthritis Clinical Trials:Arthritis and Rheumatism, June 1993, 36(6):729-740).

The patient and physician assessments of disease activity change fromthe baseline by at least 2 units, resulting from treatment with CTLA4Ig,L104EA29YIg, or placebo, are shown in FIGS. 6A and B. The responsesappear to be dose-dependent with more marked improvement for the higherdoses of active drugs.

The percent reduction in C-reactive protein (CRP) levels in patientstreated with CTLA4Ig, L104EA29YIg, or placebo, is shown in FIGS. 7A andB. The responses appear to be dose-dependent with a clear decrease forthe 2 and 10 mg/kg active treatment groups. In addition, FIG. 7B showedthat the difference is quite significant compared to placebo with 95%confidence intervals. FIG. 7C shows the changes in serum level changesfrom baseline at day 85.

The amount of serum soluble IL-2 receptor in patients treated withCTLA4Ig, L104EA29YIg, or placebo, is shown in FIG. 8. The reduction insoluble IL-2 receptor levels appears to be dose-dependent.

The amount of serum soluble ICAM-1 and soluble E-selectin in patientstreated with CTLA4Ig, L104EA29YIg, or placebo, is shown in FIG. 33. Thereduction in soluble ICAM-1 and soluble E-selectin levels appears to bedose-dependent.

The median and mean tender joint counts in patients treated with CTLA4Igor placebo over time are shown in FIGS. 9A and B. The change frombaseline (e.g., reduction in tender joints) appears to be more importantin the 2 and 10 mg/kg treated groups, than in the placebo or 0.5 mg/kggroups.

The median and mean swollen joint counts in patients treated withCTLA4Ig or placebo over time are shown in FIGS. 10A and B. The changefrom baseline (e.g., reduction in swollen joints) appears to be moreimportant in the 2 and 10 mg/kg treated groups than placebo or 0.5 mg/kggroups.

The mean pain assessment scores over time in patients treated withCTLA4Ig or placebo are shown in FIG. 11. The change from baseline (e.g.,reduction in pain) appears to be more important in the 2 and 10 mg/kgtreated groups than placebo or 0.5 mg/kg groups.

The mean disease activity assessment scores assessed by patient orphysician in patients treated with CTLA4Ig or placebo over time areshown in FIGS. 12A and B. The change from baseline (e.g., reduction indisease activity) appears to be more important in the 2 and 10 mg/kgtreated groups than placebo or 0.5 mg/kg groups.

The median and mean tender joint counts in patients treated withL104EA29YIg (denoted as LEA in the figures) or placebo over time areshown in FIGS. 13A and B. The change from baseline (e.g., reduction intender joints) appears to be dose-dependent.

The median and mean swollen joint counts in patients treated withL104EA29YIg (denoted as LEA in the figures) or placebo over time areshown in FIGS. 14A and B. The change from baseline (e.g., reduction inswollen joints) appears to be more important in the 2 and 10 mg/kgtreated groups than placebo or 0.5 mg/kg groups.

The mean pain assessment scores in patients treated with L104EA29YIg(denoted as LEA in the figures) or placebo over time are shown in FIG.15. The change from baseline (e.g., reduction in pain) appears to bedose-dependent.

The mean disease activity assessment scores evaluated by patient orphysician in patients treated with L104EA29YIg (denoted as LEA in thefigures) or placebo over time are shown in FIGS. 16A and B. The changefrom baseline (e.g., reduction in disease activity) appears to bedose-dependent.

The percent improvement of physical disability assessed by HAQ at day 85for patients treated with CTLA4Ig, L104EA29YIg, or placebo are shown inFIG. 17 (Health Assessment Questionnaire (HAQ); Fries, J. F., et al.,1982 J of Rheumatology 9:789-793). There is a clear dose dependentimprovement with this parameter.

The changes from baseline for soluble IL-2r and C-reactive proteinlevels were dose-dependent in both treatment groups. After treatment,soluble IL-2r levels were −2%, −10%, and −22% for CTLA4Ig and −4%, −18%,and −32% for L104EA29YIg at 0.5, 2.0, and 10.0 mg/kg respectively,compared to +3% for the placebo. C-reactive protein levels were +12%,−15%, and −32% for CTLA4Ig and +47%, −33%, and −47% for L104EA29YIg at0.5, 2.0, and 10.0 mg/kg respectively, compared to +20% for the placebo(FIG. 7A).

No clinically remarkable findings with respect to routine hematologytesting, chemistry laboratory testing with the exception of slightsuppressions in IgA and IgG levels at the higher doses of both drugs,physical findings, or vital signs assessments were observed. Notably,neither medication induced drug-specific antibodies.

Example 4

The following Examples describe phase II clinical studies of humanpatients that will be administered L104EA29YIg, to reduce or preventstructural damage, including bone or joint erosion using validatedradiographic scales. This improvement in reducing or preventingstructural damage is parallel to the clinical improvement measured bythe clinical parameters.

The status of the bone structure is monitored in some of the humanpatients prior to treatment with CTLA4Ig or L104EA29YIg. These patientsare administered from 0.5 to 20 mg/kg of CTLA4Ig or L104EA29YIgchronically every two to twelve weeks (alone or in combination withother agents) to maintain their therapeutic improvement over time.Radiographs of patients' hands and feet are taken at predefinedintervals: 6 months, and then yearly, as recommended by the FDAguidelines. These patients are monitored in long-term extension after 6and 12 months to determine if treatment with CTLA4Ig or L104EA29YIgreduces the progression of bone deterioration, and then yearly. Thepatients are monitored by radiographic methods, including X-ray and/ormagnetic resonance imaging (MRI), according to standard practice in theart (Larsen, A. K. and M. Eek 1977 Acta. Radiol. Diag. 18:481-491;Sharp, J. T., et al., 1985 Arthritis and Rheumatism 28:1326-1335). Theresults of the radiographic data are evaluated for prevention ofstructural damage, including slowing the progression of bone erosion andcartilage damage, with joint space narrowing and/or prevention of newerosions.

Example 5

A Study to Evaluate the Safety and Clinical Efficacy of Two DifferentDoses of CTLA4Ig Administered Intravenously to Subjects with ActiveRheumatoid Arthritis While Receiving Methotrexate

Rheumatoid Arthritis (RA) treatment is rapidly changing with anincreased willingness to use more aggressive therapies to achieve largerincreases in efficacy and higher success rates. The ultimate goal is toimprove the subject condition in a more intensive way, by raising therate of major and complete clinical response, to treatment andmaintaining this benefit with acceptable safety.

Methotrexate remains the cornerstone of the RA treatment. It was thefirst agent that demonstrated early onset of action, superior efficacyand tolerability compared to the classical DMARDs (e.g. gold,hydroxychloroquine, sulfasalazine) used to treat RA. Clinical benefitmay be seen as early as 3 weeks after initiating treatment, and themaximal improvement is generally achieved by 6 months. However,methotrexate has a number of limitations. For example, despite itsincreased tolerability, the window between efficacy and liver toxicityis quite narrow. Subjects treated with methotrexate require carefulmonitoring and unacceptable toxicity is often the reason fordiscontinuation of treatment.

Methotrexate also does not appear to efficiently control diseaseprogression or joint deterioration. For some subjects, practitionersfeel compelled to add a second DMARD with the hope of increasingefficacy despite the risk of increased toxicity. Alternatively,co-treatment with methotrexate and a costimulator blocker (e.g. CD80 andCD86 blockers such as CTLA4Ig) that target the auto-immune mechanismthat lies upstream of the cytokine inflammatory cascade, may alsoincrease efficacy.

As noted in Example 3, above, significant clinical responses andreductions in surrogate markers of disease activity were observed forCTLA4Ig at doses of 2 and 10 mg/kg with a good tolerability profile. Ithas also been confirmed that the composition CTLA4Ig, used in Example 3above, did not induce any side effects. As a result, it was decided tocontinue the clinical development of CTLA4Ig for rheumatoid arthritis inPhase IIB.

The following provides a description of a Phase IIB clinical study ofhuman patients administered soluble CTLA4 molecule with methotrexate,and the results of the study after six months.

This Example describes a twelve month study in which primary efficacywas assessed after all subjects completed six months of treatment ordiscontinue therapy. Efficacy, safety, and disease progression were alsoassessed throughout the duration of the study.

The study utilized a randomized, double blind, placebo controlled,parallel dosing design. The study was designed to evaluate the safety,clinical activity, immunogenicity and pharmacokinetics of two doses ofCTLA4Ig: 2 or 10 mg/kg. A total of approximately 330 subjects withactive RA and receiving methotrexate were randomized to 1 of 3 dosingarms: CTLA4Ig at 2 mg/kg (N=110), 10 mg/kg (N=110) and placebo controlgroup (N=110) given monthly infusions for 12 months. All groupscontinued on weekly methotrexate treatment (10-30 mg weekly) (FIGS.57-62).

CTLA4Ig or a placebo were also administered on Day 15. Each dose ofstudy medication was infused intravenously over approximately 30minutes. The primary efficacy endpoint was the ACR 20 response rateafter 6 months.

For the first 6 months, subjects were not allowed to alter their dosesof corticosteroids, glucocorticoids or NSAIDs. Increases in methotrexatewere also not permitted during the first six months. Decreases inmethotrexate were permitted only if it was felt to be causing toxicity.Subjects were treated with methotrexate for at least 6 months, and at astable dose for 28 days prior to first treatment of CTLA4Ig or placebo.DMARDs other than methotrexate were not permitted. Low-dose stablecorticosteroids use (at 10 mg daily or less) and/or use of stablenon-steroidal anti-inflammatory drugs (NSAIDs), including acetylsalicylic acid (ASA), was allowed. Analgesics that did not contain ASAor NSAIDs were permitted in subjects experiencing pain not adequatelycontrolled by the baseline and study medications, except for 12 hoursbefore a joint evaluation. Decreases in NSAIDs were permitted but onlyif due to adverse events such as gastrointestinal toxicity.

Test Product, Dose and Mode of Admistration, Duration of Treatment

CTLA4Ig at 2 mg/kg or 10 mg/kg was infused every two weeks for the firstmonth, and monthly thereafter for 12 months.

All subjects received weekly doses of methotrexate (10-30 mg) for atleast six months prior to randomization and maintained at the entry dosefor the first 6 months of the trial. Doses could only be decreased fortoxicity during the first six months.

Criteria for Evaluation

The primary endpoint of the first stage of the study was the proportionof subjects meeting the American College of Rheumatology criteria for20% improvement (ACR 20) on Day 180 (month six). The ACR 20 definitionof improvement is a 20% improvement from baseline in the number oftender and swollen joint counts, and a 20% improvement from baseline in3 of the following 5 core set measurements: subject global assessment ofpain, subject global assessment of disease activity, physician globalassessment of disease activity, subject assessment of physical functionand acute phase reactant value (C-reactive protein (CRP)). Theevaluation for 50% improvement (ACR 50) and 70% improvement (ACR 70)follow similarly. Subjects who discontinued the study due to lack ofefficacy (i.e. worsening RA) were considered as ACR non-responders fromthat time on. For all subjects who dropped out for other reasons, theirACR response at the time of discontinuation was carried forward.

Statistical Methods

Two doses of CTLA4Ig (2 mg/kg and 10 mg/kg) were compared with theplacebo control group. All subjects were maintained at the same stableentry doses of methotrexate. The primary analysis was the comparison ofCTLA4Ig 10 mg/kg with placebo. Sample sizes were based on a 5% level(2-tailed) of significance. Based on published studies, the placebo plusmethotrexate control ACR 20 response rate at 6 months is about 25%. Asample of 107 subjects (adjusted for a possible 15% dropout) pertreatment arm was determined to yield a 94% power to detect a differenceof 25% at the 5% level (two-tailed). Similarly, the sample wasdetermined to yield a power of 95% and 90% to detect differences of 20%and 14% in ACR 50 and ACR 70, respectively. If the comparison betweenCTLA4Ig 10 mg/kg and placebo was significant with regards to ACR 20,then the comparison between CTLA4Ig 2 mg/kg and placebo was carried out.This second testing should have a power of 88%. This sequentiallyrejective procedure based on Chi-square tests was also used to test fordifferences in ACR 50 and ACR 70 responses.

All efficacy analyses were based on a data set containing all availableassessments from all subjects who received at least one dose of studymedication.

Percent changes from baseline were also reported for the individualcomponents of the ACR. For subjects who discontinued, their lastobservation was carried forward.

Results

Demography and Baseline Characteristics

TABLE III Subject Disposition and Demographics Methotrexate +Methotrexate + CTLA4Ig CTLA4Ig Methotrexate + 10 mg/kg 2 mg/kg PlaceboEnrolled/Randomized 115 105 119 Completed 99 (86.1%) 82 (78.1%) 78(65.5%) Discontinued 16 (13.9%) 23 (21.9%) 41 (34.5%) Adverse Events 2(1.7%) 7 (6.7%) 7 (5.9%) Lack of Efficacy 12 (10.4%) 13 (12.4%) 29(24.4%) Other 2 (1.7%) 3 (2.9%) 5 (4.2%) Age (yrs) - Mean (Range) 55.8(17-83) 54.4 (23-80) 54.7 (23-80) Weight (kg) - Mean (Range) 77.8(40.1-144) 78.7 (48.4-186.8) 79.9 (44-140) Sex 75% females 63% females66% females Race 87% white 87% white 87% white Duration of Disease (yrs)9.7 ± 9.8 9.7 ± 8.1 8.9 ± 8.3 Mean ± SD

Demographic and baseline clinical characteristics were similar among thetreatment groups. Sixty three to 75 percent of subjects were female, 87%were Caucasian. The mean duration of the disease at entry was 9.7±9.8,9.7±8.1, and 8.9±8.3 years respectively in the 10, 2 mg/kg and thecontrol group. The mean weight in kg was very similar between 77.8 and79.9 kg with a range of 40.1 to 186.8 kg (Table III).

After 6 months, more subjects had discontinued from the control group(35.5%) than from the active treatment groups; 13.9% and 21.9% for the10 and 2 mg/kg treated groups, respectively. The main reason was lack ofefficacy: with 24.3% discontinuing in the control group, as opposed to12.4% and 10.4% discontinuing in the 2 and 10 mg/kg groups,respectively. The discontinuation rate due to adverse events was lowerin the 10 mg/kg group with 1.7%, while it was 6.7% and 5.9% in the 2mg/kg and the control groups, respectively.

During the first 3-4 months, the discontinuations appeared at a fasterrate in the control group compared to the active-treatment groups. AfterDay 120, the discontinuations for all treatment groups stabilized forthe duration of the primary treatment period (six months).

TABLE IV Baseline Clinical Characteristics Methotrexate + Methotrexate +CTLA4Ig CTLA4Ig Methotrexate + 10 mg/kg 2 mg/kg Placebo (n = 115) (n =105) (n = 119) Tender Joints 30.8 ± 12.2 28.2 ± 12.0 29.2 ± 13.0 (mean ±SD) Swollen Joints 21.3 ± 8.4  20.2 ± 8.9  21.8 ± 8.8  (mean ± SD) Pain(VAS 100 mm) 62.1 ± 21.4 64.5 ± 22.3 65.2 ± 22.1 (mean ± SD) PhysicalFunction 1.0 ± 0.5 1.0 ± 0.5 1.0 + 0.6 (MHAQ score of 0 to 3)(mean ± SD)Subject global 60.1 ± 20.7 59.4 ± 23.7 62.8 ± 21.6 assessment (VAS 100mm) (mean ± SD) Physician global 62.1 ± 14.8 61.0 ± 16.7 63.3 ± 15.5assessment (VAS 100 mm) (mean ± SD) CRP (mg/dL) 2.9 ± 2.8 3.2 ± 2.6 3.2± 3.2 Morning Stiffness 97.9 ± 63.1 104.1 ± 63.9  106.0 ± 64.2  (inmin.)

The mean number of tender and swollen joints at baseline was comparableamong the three treatment groups. The mean number of tender joints andswollen joints in the 10 mg group was 30.8±12.2 and 21.3±8.4,respectively. The mean number of tender joints and swollen joints in the2 mg group was 28.2±12.0, and 20.2±8.9, respectively. The mean number oftender joints and swollen joints in the control group was 29.2±13.0, and21.8±8.8, respectively. These assessments and all other clinicalassessments were similar among all treatment groups (Table IV).

ACR Responses and Core Components

TABLE V ACR Response at 6 months Methotrexate + Methotrexate + CTLA4IgCTLA4Ig Methotrexate + 10 mg/kg 2 mg/kg Placebo (n = 115) (n = 105) (n =119) ACR 20 60.0% 41.9% 35.3%   Difference from 24.7 6.6 — control group95% CI 11.9, 37.5 −6.2, 19.4 — p-value <0.001 0.31 — ACR 50 36.5% 22.9%11.8%   Difference from 24.8 11.1 — control group 95% CI 13.8, 35.7 1.2,20.9 — p-value <0.001 0.027 — ACR 70 16.5% 10.5% 1.7%  Difference from14.8 8.8 — control group 95% CI 7.5, 22.2 2.7, 14.9 — p-value <0.0010.005 —

The improvements in ACR 20, 50, and 70 response rates in the 10 mg/kgtreatment group, at six months relative to the methotrexate controlgroup, were statistically significant (FIGS. 34-38, 40). Theimprovements in ACR 50, and ACR 70 for the 2 mg/kg group were alsostatistically significant. The difference in ACR 20 response between the2 mg/kg group and the control group was 6.6%. This difference was notstatistically significant, p=0.31 (Table V, FIG. 49).

FIGS. 34-37 presents the ACR response rates from Day 1 to Day 180. FIGS.38 and 40 presents the ACR20, -50 and -70 response rates on day 180 forthe various treatment groups. The ACR 50 and ACR 70 response ratessuggest the possibility that maximal efficacy may not have been achievedat 10 mg/kg.

FIG. 39 shows the proportion of new tender and swollen joints at day 180of the study after therapy with methotrexate alone or in combinationwith CTLA4Ig (administered at 2 or 10 mg/kg body weight of subject).

FIG. 46 shows the mean percent improvement in physical function frombaseline as measured by HAQ.

TABLE VI Individual ACR Components at Day 180 (Mean Percent Improvement)Methotrexate + Methotrexate + CTLA4Ig CTLA4Ig Methotrexate + 10 mg/kg 2mg/kg Placebo Core Components (n = 115) (n = 105) (n = 119) TenderJoints 59.9% 43.3% 32.1% Swollen Joints 54.9% 45.1% 33.4% Pain 46.4%22.7%  8.4% Physical Function 41.5% 17.3% 14.1% (mHAQ) Subject global40.8%  9.6% 17.6% assessment Physician global 52.0% 38.6% 25.6%assessment CRP 31.5% 16.2% −23.6%  

The 2 and 10 mg/kg dose groups demonstrated some degree of efficacyamong all clinical components of the ACR response criteria (Table VI;FIGS. 41-45, 47-48); the subject's global assessment in the 2 mg/kg dosegroup being the only exception. The reduction of tender and swollenjoints appears dose-dependent. The number of tender joints was decreasedby 59.9%, 43.3% and 32.1% in the 10 mg/kg, 2 mg/kg and control groups,respectively. A similar pattern was observed for the swollen jointcounts with a decrease of 54.9%, 45.1% and 33.4% in the 10 mg/kg, 2mg/kg and control groups, respectively. The greatest differencesrelative to the control group were observed with the pain assessmentwhich decreased 46.4% and 22.7% relative to baseline for 10 mg and 2mg/kg CTLA4Ig, respectively, compared to 8.4% in the control group. Themean CRP decreased 31.5% and 16.2% relative to baseline in the 10 and 2mg/kg groups compared to an increase of 23.6% in the control group.

Health-Related Quality of Life

The impact of CTLA4Ig on health-related quality of life (HRQOL) wasmeasured by the Medical Outcomes Study Short Form-36 (SF-36). The SF-36was administered to all subjects at baseline, 90 and 180 days. The SF-36consists of 36 items which covers eight domains (physical function,role-physical, bodily pain, general health, vitality, social function,role emotional, and mental health). These individual domains are used toderive the physical and mental component summary scores which range from0 to 100, with higher scores indicating better quality of life. Absolutedifferences of 5 or more in the SF-36 scores were considered clinicallymeaningful.

Compared to subjects treated with placebo, subjects in the CTLA4Ig 10mg/kg group also experienced statistically significantly greaterimprovement in all 8 domains of the SF-36 (FIG. 50-51). For subjectstreated with CTLA4Ig 2 mg/kg, the improvements were also greater thanthose treated with placebo, but the differences were not statisticallysignificant (FIG. 50-51).

Baseline SF-36 scores were comparable between the three treatmentgroups. Improvements in quality of life show a clear dose-response trendafter 6 months of treatment. Subjects in the CTLA4Ig 10 mg/kg treatmentgroup demonstrated clinically and statistically significant improvementsfrom baseline in all 8 domains of the SF-36. The greatest effects wereshown in the role-physical, bodily pain, and role-emotional domains.This positive finding was consistent with the efficacy results. Forsubjects treated with CTLA4Ig 2 mg/kg, improvements from baseline werealso statistically significant for all domains except mental health.

Pharmacokinetics

Pharmacokinetic Parameter Values CMAX TMAX AUC(TAU) T-HALF CLT VSS(μG/ML) (H) (μG · H/ML) (Days) (ML/H/KG) (L/KG)  2 mg/kg MEAN 57.960.50* 10176.14 13.50 0.23 0.07 SD 16.93 (0.00, 4.00) 3069.84 5.91 0.130.04 N 15 15 15 15 15 15 10 mg/kg MEAN 292.09 0.50* 50102.56 13.11 0.220.07 SD 67.78 (0.00, 4.00) 15345.95 5.32 0.09 0.03 N 14 14 14 14 14 14*Median (minimum, maximum)

The pharmacokinetics of CTLA4Ig were derived from serum concentrationversus time data between dosing days 60 and 90. Samples were collectedprior to dosing on day 60, at 0.5, and 4 h after dosing, on days 67, 74,81, and prior to dosing on Day 90. The preliminary data indicate thatboth Cmax and AUC values increase in a proportion comparable to the doseincrement. For nominal doses increasing in a 1:5 proportion, both theCmax and AUC values increased in the proportion of 1:5.04 and 1:4.92,respectively. T-HALF, CLT, and Vss values appeared to be comparable anddose independent.

Mean Vss values were 0.07 L/kg for both dose levels, which wasapproximately 1.6-fold the plasma volume.

Pharmacodynamics

TABLE VII Mean Baseline Values for Pharmacodynamic BiomarkersMethotrexate + Methotrexate + CTLA4Ig CTLA4Ig Methotrexate + 10 mg/kg 2mg/kg Placebo Biomarker (n = 115) (n = 105) (n = 119) CRP (mg/dL) 2.93.2 3.2 RF (IU/L) 207 274 179 IL-2r (pg/ml) 1388 1407 1398 IL-6 (pg/ml)26.7 31.7 21.4 TNFα (pg/ml) 11.8 6.0 11.9

Serum levels of pharmacodynamic biomarkers were analyzed at varioustimes during the study. Baseline values are shown in Table VII. Thevalues on Day 180 relative to baseline are shown in the FIGS. 52-56.

CRP levels decreased from baseline in both CTLA4Ig-treated groups morethan in the control group, with greater reduction observed in the 10mg/kg dosing group (see FIGS. 47, 48 and 52).

Rheumatoid factor levels decreased from baseline in both CTLA4Ig-treatedgroups more than in the control group, with greater reduction observedin the 10 mg/kg dosing group (see FIG. 53).

Soluble IL-2r levels decreased from baseline in both CTLA4Ig-treatedgroups more than in the control group, with greater reduction observedin the 10 mg/kg dosing group (see FIG. 54).

Serum IL-6 levels decreased from in both CTLA4Ig-treated groups morethan in the control group (see FIG. 55).

The effects of CTLA4Ig on serum TNFa levels were inconclusive. The 2mg/kg group increased and the 10 mg/kg group decreased relative to thecontrol group (see FIG. 56).

Safety

CTLA4Ig was well tolerated at all doses. There were no deaths,malignancies or opportunistic infections in any subjects receivingCTLA4Ig. Serious adverse events (SAEs) and non-serious adverse events(NSAEs) were similar or less frequent in the active-treatment groupscompared to the control group.

Fewer subjects in the 10 mg/kg group discontinued due to adverse eventsrelative to the control group (1.7% vs 5.9%, respectively). Thediscontinuations due to adverse events in the 2 mg/kg were similar tothe control group (6.7% vs 5.9%, respectively). The SAEs followed apattern similar to the discontinuations due to adverse events.

No serious adverse events in the 10 mg/kg dose group were consideredrelated to the study drug.

Immunogenicity

No anti-drug antibody responses were detected through Day 180 at bothdose levels of CTLA4Ig.

CTLA4Ig significantly reduced the signs and symptoms of rheumatoidarthritis in subjects receiving methotrexate as assessed by ACR responsecriteria. The effects of CTLA4Ig appear to increase in proportion todose level. The improvement from baseline in all ACR core components ishigher in the 10 mg/kg group than the 2 mg/kg group. CTLA4Ig at 10 mg/kgdoses demonstrated clinically and statistically significant improvementsin all 8 domains of the SF-36. All pharmacodynamic biomarkers assayedappeared to decrease in proportion to CTLA4Ig dose level except forTNFα. CTLA4Ig was safe and well tolerated in subjects with rheumatoidarthritis receiving methotrexate. The adverse event profile for bothCTLA4Ig doses was similar to the control group.

Example 6

A Study of a Co-Stimulation Blocker, CTLA4Ig, Given Monthly inCombination with Etanercept to Patients with Active Rheumatoid Arthritis

The following Example provides a description of the administration ofCTLA4Ig, in combination with etanercept, to treat patients with activeRheumatoid Arthritis.

Etanercept, along with infliximab, comprises a new generation ofRheumatoid Arthritis drugs which targets Tumor Necrosis Factor (TNF.Etanercept is a dimeric fusion protein having an extracellular portionof the TNF receptor linked to the Fc portion of human immunoglobulin(IgG1). This fusion protein binds to TNF, blocks its interactions withcell surface TNF receptors and render TNF molecules biologicallyinactive.

This example describes a twelve month study in which efficacy wasassessed after all subjects completed six months of treatment ordiscontinued therapy. Efficacy, safety and disease progression were alsoassessed throughout the duration of the study.

The study utilized a randomized, double-blind, placebo controlled,parallel dosing design. A total of approximately 141 subjects withactive RA and receiving etanercept (25 mg twice weekly) were randomizedto 1 of 2 dosing groups: 1) a group receiving CTLA4Ig at 2 mg/kg (n=94)plus etanercept or 2) a placebo group receiving etanercept only (n=47).

Test Product, Dose and Mode of Administration, Duration of Treatment

All subjects received etanercept (25 mg twice weekly) for at least 3months prior to treatment.

Infusions of CTLA4Ig were given on Days 1, 15, 30, and monthlythereafter, for 6 months (primary treatment phase). Each dose of studymedication was infused intravenously for approximately 30 minutes.

The primary treatment phase of the study took place during the first sixmonths of treatment. During this period, subjects were required toremain on stable doses of etanercept (25 mg twice weekly). DMARDs otherthan etanercept were not permitted. Low-dose stable corticosteroid (at10 mg daily or less) and/or stable non-steroidal anti-inflammatory drug(NSAID), including acetyl salicylic acid (ASA), use was allowed.Analgesics (that do not contain ASA or NSAIDs) were permitted insubjects experiencing pain that was not adequately controlled by thebaseline and study medications, except for 12 hours before a jointevaluation.

Criteria for Evaluation

The primary endpoint of this study was to collect data regarding theproportion of subjects meeting modified American College of Rheumatology(ACR) criteria for 20% improvement (ACR 20) after six months. Themodified ACR 20 criteria were used to accommodate the low CRP levels inthis study's subject population. The modified ACR criteria were definedas 1) a greater than 20% improvement in tender and swollen joint countand 2) a greater than 20% improvement in 2 of the remaining 4 core dataset measures (global pain, physician, subject, functional assessment).CRP, which is normally a part of the standard ACR core data sets, wasnot included in the modified ACR criteria due to the low levels of CRPin subjects using TNF blockers, such as etanercept. The standard ACRcriteria, and two alternative criteria (SF-36 Physical Health and SF-36Mental Health) were also evaluated as secondary endpoints.

Statistical Methods

Treatment of a group of patients with CTLA4Ig 2 mg/kg in combinationwith etanercept was compared with a control group treated with placeboplus etanercept. Based on previous studies with etanercept in similarpatient populations, it was assumed that the modified ACR 20 responserate (modified criteria for evaluation) at 6 months would be 35% in thecontrol group. This is the rate of response expected among subjects whodid not respond adequately to etanercept therapy. Using a 2:1randomization, a sample of 141 (adjusted for a possible 10% dropout)subjects (47 control/94 CTLA4Ig) yields a 90% power to detect adifference of 30% at the 5% level of significance (2-tailed, based on achi-square test with no adjustment for continuity correction).

Similarly, the sample was determined to yield a power of 91% and 83% todetect differences of 30 and 25% in ACR 50 and 70, respectively.However, due to slow enrollment, only 122 subjects were randomized and121 treated and analyzed (one subject was randomized but never receivedtreatment).

Demography and Baseline Characteristics

TABLE 1 Subject Disposition at Day 180 CTLA4Ig + Placebo + etanerceptetanercept TOTAL Randomized* 85 36 121 Completed 68 (80%) 22 (61%) 90(74%) Discontinued 17 (20%) 14 (39%) 31 (26%) Adverse Events 6 (7.0%) 1(2.7%) 7 (6%) Lack of Efficacy 6 (7.0%) 12 (33%) 18 (15%) Other 5 (5.8%)1 (2.7%) 6 (5%) *Excludes one subject that did not receive treatment.

After six months, the proportion of total discontinuations were higher(39%) in the placebo plus etanercept treatment group compared to theCTLA4Ig plus etanercept group (20%). The difference was driven by ahigher rate of discontinuation due to lack of efficacy in the placeboplus etanercept group (Table 1).

Demographic characteristics were similar between treatment groups. Themajority of subjects were female and Caucasian. The mean duration of thedisease was 13 years and the mean age was 52 years (Table 2).

TABLE 2 Mean Baseline Demographic and Clinical Characteristics CTLA4Ig +Placebo + etanercept etanercept TOTAL N = 85 N = 36 N = 121 Mean Age:yrs 50 (24-74) 55 (28-72) 52 (24-74) (Range) Mean Weight: kg 81 (45-154)79 (46-126) 81 (45-154) (Range) Gender: female: 66 (78%) 26 (72%) 92(76%) n (%) Race: Caucasian - 80 (94%) 36 (100%) 116 (96%) n (%) MeanDuration of 13.0 ± 10.1 12.8 ± 8.6  13.0 ± 9.7 Disease: yrs ± sd TenderJoints 28.7 ± 14.0 29.5 ± 13.7  28.9 ± 13.8 (out of 68) - mean ± sdSwollen Joints - 19.6 ± 9.4  20.3 ± 11.0 19.8 ± 9.9 (out of 66) - mean ±sd

Baseline clinical characteristics were similar between treatment groupsincluding a mean of 29 tender joints and 20 swollen joints. With theexception of CRP values, which were lower, the baseline characteristicswere typical of subjects with active rheumatoid arthritis and enrolledin clinical studies (Table 2).

ACR Responses and Core Components

The improvements in the ACR 20 and ACR 70 responses in theCTLA4Ig+etanercept group were statistically significant compared to theCTLA4Ig+placebo group (Table 3 and FIG. 63).

TABLE 3 Modified ACR Response at Day 180 - number of subjects (%)* ACR20 ACR 50 ACR 70 CTLA4Ig + 48.2% 25.9% 10.6% etanercept*** Diff. fromPlacebo + 20.5%  6.4% 10.6% etanercept 95% CI (1.2, 39.7) (−10.2, 23.1)(0.4, 20.8) p-Value  0.037**  0.448  0.042** *See Criteria forEvaluation **p < 0.05 (probability for ACR response in CTLA4Ig +etanercept vs. placebo + etanercept) ***N = 85 and N = 36 for CTLA4Ig +etanercept: and Placebo + etanercept, respectively

By two months of treatment, numerically higher responses on allcomponents of the ACR criteria were observed for the CTLA4Ig plusetanercept group. Three of the seven ACR components are shown in FIG.64A-C.

The mean improvements in the individual components of the ACR criteriaon Day 180 were consistently greater in the CTLA4Ig plus etanercepttreatment group compared to the placebo plus etanercept group (Table 4).

TABLE 4 Mean Percent (SE) Improvement in Individual ACR Components atDay 180 CTLA4Ig + Placebo + etanercept etanercept ACR Component N = 85 N= 36 Tender Joints 42% (5.5) 24% (8.3) Swollen Joints 37% (5.0) 21%(8.1) Pain 34% (4.3) −1% (10.8) Physical Function (MHAQ) 31% (5.2) −5%(13.8) Subject Global Assessment 27% (5.4) 3% (9.5) Physician GlobalAssessment 43% (4.3) 27% (5.8)Quality of Life

Compared to baseline, subjects in the CTLA4Ig plus etanercept groupdemonstrated statistically significant improvements at Day 180 in all 8subscales of the SF-36—compared to only one (physical function) insubjects in the placebo plus etanercept group. The absolute changes inHRQOL subscales were considered clinically meaningful.

Compared to the placebo plus etanercept group, subjects in the CTLA4Igplus etanercept group experienced statistically significantly greaterimprovement in 4 subscales of the SF-36: role-physical, bodily pain,vitality, and social function (FIG. 65). Improvements in the other 4subscales were also greater than the placebo plus etanercept group,although they were not statistically significant.

Safety

No deaths or opportunistic infections occurred during the first sixmonths of this study. Among the most frequently reported adverse events,headache, upper respiratory infection, musculo/skeletal pain,nausea/vomiting, hypertension, and diarrhea occurred at a higher rate inthe CTLA4Ig plus etanercept group compared to the placebo plusetanercept group. Sinus abnormalities and rash were slightly higher inthe CTLA4Ig plus etanercept group, as well.

More subjects in the CTLA4Ig plus etanercept group experienced seriousadverse events (SAE) (7.1%) than the etanercept plus placebo group(2.8%). However, no SAEs were considered related to the study drug.

Two subjects receiving CTLA4Ig and etanercept had a dermatologicalmalignancy. One subject had a basal cell carcinoma that was excisedafter the Day 150 visit. The other subject had a squamous cell carcinomawhich was a pre-existing lesion that the subject decided to have removedafter the Day 120 visit. Another subject experienced angioedema that wasconsidered by the investigator to be a drug reaction to azithromycin.

All adverse events (AEs) leading to discontinuation were of either ofmild or of moderate intensity. One discontinuation in the CTLA4Ig plusetanercept group, due to a tremor, was considered a serious adverseevent.

Immunogenicity

No subjects receiving CTLA4Ig seroconverted for CTLA4Ig or CTLA4-Tspecific antibodies. No significant change in GMTs for CTLA4Ig orCTLA4-T specific antibodies was observed.

Comparison Between CTLA4Ig/Etanercept and CTLA4Ig/Methotrexate ACRResponses

TABLE 5 CTLA4Ig + etanercept vs. CTLA4Ig + methotrexate ACR responses (%improvement): CTLA4Ig + Etanercept^(a) CTLA4Ig + Methotrexate^(b)(IM101-101) (IM101-100) 2 mg/kg 0 mg/kg^(c) 10 mg/kg 2 mg/kg 0 mg/kg^(c)N = 85 N = 36 N = 115 N = 105 N = 119 ACR 20 48.2%^(d) 27.8% 60.0%^(d)41.9%  35.3% ACR 50 29.3%  19.4% 36.5%^(d) 22.9%^(d) 11.8% ACR 7010.6%^(d)   0% 16.5%^(d) 10.5%^(d)  1.7% ^(a)Modified ACR. See criteriafor evaluation ^(b)Standard ACR criteria ^(c)Placebo + Backgroundtherapy (etanercept or methotrexate) ^(d)p < 0.05 for the difference inACR response vs placebo + background therapy

The efficacy of CTLA4Ig plus etanercept at 2 mg/kg was similar to thatobserved in subjects receiving the same dose of CTLA4Ig plusmethotrexate therapy (Example 5). However, the criteria for evaluationin the methotrexate (Example 5) trial was the standard ACR, thatincludes CRP among the core components, while in the etanercept trial(Example 6) the criteria for evaluation was the modified ACR, thatexcludes CRP.

Conclusion

Preliminary assessment of the study at six months found that CTLA4Ig (2mg/kg) in combination with etanercept reduced the signs and symptoms ofrheumatoid arthritis, as compared with etanercept alone. The increasesin the modified ACR20 and ACR 70 assays were statistically significant.Efficacy of CTLA4Ig plus etanercept therapy was observed within onemonth of the start of treatment. CTLA4Ig was generally safe and welltolerated when administered in combination with etanercept with thesafety profile similar to etanercept therapy alone. CTLA4Ig was notimmunogenic during the six month trial period. Additionally, theefficacy of CTLA4Ig therapy in combination with etanercept (Example 6)was similar to the same dose of CTLA4Ig with methotrexate (Example 5).

Example 7

One-Year Results of a Phase IIB, Multicenter, Randomized, Double-Blind,Placebo-Controlled Study to Evaluate the Safety and Clinical Efficacy ofTwo Different Doses of BMS-188667 Administered Intravenously to Subjectswith Active Rheumatoid Arthritis While Receiving Methotrexate

The following Example provides the one-year results from a Phase IIB,multi-center, randomized, double-blind, placebo controlled clinicalstudy to evaluate the safety and clinical efficacy of administering twodifferent doses of CTLA4Ig in combination with methotrexate to treatpatients with active Rheumatoid Arthritis (RA). The study presented inthis example is a continuance of the six-month study presented inExample 5.

Based on preliminary efficacy results from Example 3, supra, and thestandard practice of adding other therapies to MTX in the treatment ofRA, this study was designed to test the hypothesis that CTLA4Ig(BMS-188667) combined with MTX may have greater clinical efficacy whencompared with MTX plus placebo in RA subjects who still have activedisease despite MTX treatment.

The results presented in this clinical study report are based on datafrom an analysis performed after all subjects completed 6 months oftreatment and again after all subjects completed 12 months of treatment.

Throughout this Example, the 10 mg/kg CTLA4Ig plus MTX group may bereferred to as the 10 mg/kg group, the 2 mg/kg plus MTX group isreferred to as the 2 mg/kg group, and the CTLA4Ig (BMS-188667) placeboplus MTX group is referred to as the placebo group.

Study Methodology

This study compared the clinical efficacy of two different doses (10 and2 mg/kg) of CTLA4Ig (BMS-188667) combined with methotrexate (MTX) orwith MTX plus placebo in subjects with active RA as assessed by ACR at 6month and 12 month intervals. This study enrolled adult subjects withactive RA who had had an inadequate response to MTX.

Results after one-year of monitoring subjects with active rheumatoidarthritis who were intravenously administere: 1) CTLA4Ig at a dosage of2 mg/kg body weight with methotrexate, 2) CTLA4Ig at a dosage of 10mg/kg body weight with methotrexate, or 3) a placebo with methotrexate(hereinafter known as placebo), are presented herein.

Subjects with active RA, despite treatment with MTX and who met theinclusion/exclusion criteria for this study were randomized 1:1:1 toreceive one of the following treatments on a background of MTX therapy:CTLA4Ig (BMS-188667) 10 mg/kg, CTLA4Ig (BMS-188667) 2 mg/kg, or placebo.Subjects must have been treated with MTX (10 mg to 30 mg weekly) for atleast 6 months, at a stable dose for 28 days prior to Day 1.

Treatment Groups: Subjects were randomized 1:1:1 to one of threetreatment groups:

-   1) Group 1: CTLA4Ig (BMS-188667) 10 mg/kg by intravenous infusion-   2) Group 2: CTLA4Ig (BMS-188667) 2 mg/kg by intravenous infusion-   3) Group 3: CTLA4Ig (BMS-188667) placebo by intravenous infusion

Infusion doses were based upon the subject's body weight from thepre-treatment visit immediately prior to the Day 1 visit (for a subjecton MTX monotherapy, the weight was obtained at the screening visit; fora subject on MTX combination therapy [in combination with other DMARDs],the weight was obtained from the washout visit, Day −2). The infusiondoses were not modified during Day 1 to Day 360.

Infusions were to occur at approximately the same time of day throughoutthe study. All doses of study medication were administered in a fixedvolume of 75 mL at a constant rate over approximately 30 minutes. Theintravenous bag and line were flushed with 25 mL of dextrose 5% in water(D5W) solution at the end of each infusion. All intravenous infusionswere administered with the subject in the seated position. Subjects wereobserved for Adverse Events (Aes) and changes in vital signs (bloodpressure, heart rate, body temperature) from the start of each infusion(pre-dose, 15, 30, 45, 60, 75, 90, 120 minutes) and for a minimum of 2hours after the start of the infusion. The observation period could beextended, if clinically indicated.

During the primary phase (Day 1 to Day 180) of the study, concomitantadministration of selected medications was allowed. The permittedmedications included:

-   -   MTX: Continued use of current dose (no increases, and decreases        only for toxicity)    -   Systemic (non-topical) corticosteroids: Provided that the dose        was stable and the total dose was less than or equal to the        equivalent of prednisone 10 mg/day. Intra-articular injections        were to be avoided; however, if necessary, up to two        intra-articular injections were permitted. NOTE: A joint that        received an intra-articular injection was counted as “active” in        ALL subsequent assessments/evaluations.    -   NSAIDs, including ASA: Provided the dose was stable    -   Acetaminophen, combination products including acetaminophen and        narcotic analgesics (i.e., acetaminophen with codeine phosphate,        acetaminophen with propoxyphene napsylate, acetaminophen with        oxycodone hydrochloride, acetaminophen with oxycodone        bitartrate, etc.), or tramadol: For subjects experiencing pain        not adequately controlled by baseline or study medication        (except for 12 hours before a joint evaluation)

Table 1 is a schedule of study procedures and evaluations.

TABLE 1 Schedule of Study Procedures and Evaluations Treatment PeriodPretreatment (Day) Screen (−28 Treatment Day^(e,f,j,h) Visit Day to −2)(−2) 1 15 30 60 90 120 150 180 210 240 270 300 330 360 Screeningassessments Informed consent X Complete History and Physical X X^(i) CXRX^(a) ECG X^(a) X Stabilize/Withdraw X prohibited medications (ifnecessary)^(b) Enroll Subject X X^(m) Randomize Subject^(k) X Dosing^(b)X X X X X X X X X X X X X Interim Assessments^(f) Duration of morningstiffness X X X X X X X X X X X X Interim History and Physical X X X X XX X X X X Tender joint count X X X X X X X X X X X X X Swollen jointcount X X X X X X X X X X X X X Subject's assessment of pain X X X X X XX X X X X X Subject's global assess of X X X X X X X X X X X X diseaseactivity Physician's global assess of X X X X X X X X X X X X diseaseactivity Subject's assess of physical X X X X X X X X X X X X functionShort form-36 health X X X X X questionnaire (SF-36) Subjects responseto therapy X X X Safety Assessments Adverse event monitoring X X X X X XX X X X X X X X^(o) Weight^(g) X X X Mammogram (females only)^(l) X XVital signs X X X X X X X X X X X X X X X Labs CBC X X X X X X X X X X XX X X X Chemistry panel X X X X X X X X X X X X X X X Urinalysis X XUrine/serum pregnancy test^(d) X X X X X X X X X X X X X X X Hepatitis Bsurface antigen X Hepatitis C antibody X Pharmacodynamics (PD)Rheumatoid factor X X X X CRP X X X X X X X X X X X X X IL-2R X X X X XX X X X X Exploratory cytokines (ICAM-1 X X X X e-Selectin, IL-6 andTNFα) Pharmacokinetics X X X X Immunoglobulin determinationsQuantitative immunoglobulins X X X (IgG, IgA, IgM) ImmunogenicityAnti-BMS-188667Ab testing X X X X X X Radiographic assessments^(n)X-rays (hands/wrists and feet) X X X ^(a)Chest X-ray and ECG wasperformed if not performed within 6 months or not on file. ^(b)Ifsubject was being treated with DMARDs on top of methotrexate therapy anddid not meet initial entry criteria, the DMARDs must have been washedout for at least 28 days prior to Day 1. ^(c)This visit was requiredonly if the subject was on MTX therapy. ^(d)Urine or serum pregnancytest performed within 48 hours prior to dosing, for all women of childbearing potential. Serum pregnancy test was to be processed locally.^(e)Subjects who discontinued must have had an “early termination”visit. Assessments at this visit were identical to assessments performedon Day 360. The assessments for this visit replaced what might have beenscheduled on the day of discontinuation. Changes in current DMARD,steroid, or NSAIDs therapy were not permitted until after theseassessments were performed. Subjects were to be contacted 30 days afterdiscontinuation to capture safety data (adverse events). ^(f)Everyeffort must have been made to insure the same evaluator completed theassessment for each subject. ^(g)Most recent weight should have beenused to calculate study drug dosage. All doses administered during thestudy were be based on this weight. ^(h)For Day 15, a +/− 3 day visitwindow was permitted. For subsequent visits, a +/− 7 day visit windowwas permitted. ^(i)Complete physical examination only. ^(j)Allassessments should have been performed or administered prior to studydrug administration unless otherwise indicated. ^(k)The results of allassessments must have been reviewed for eligibility requirements beforecontacting the Central Randomization System for randomization. ^(l)SeeSection 2.1.4.3 of the protocol for mammography rationale. If notperformed within 6 months (documentation must be on file) prior tosigning consent. Subjects who discontinued from the study after Day 1required a follow-up mammogram on the one year anniversary of themammogram that was performed during the screening period. ^(m)Subject'sbody weight was provided to central randomization system. ^(n)Noradiographic assessments were required at the termination visit forsubjects who discontinued within the first nine months of treatment.^(o)Subjects who were terminated early had adverse events andconcomitant medications recorded 30 and 60 days after the last dose ofstudy medication.Efficacy AssessmentsClinical Measurements and Responses

Clinical response was assessed using the American College ofRheumatology (ACR) Core Data Set and Response Definitions. For thisassessment, data were collected on seven components: 1) tender jointcount (standardized 68 joint count); 2) swollen joint count(standardized 66 joint count); 3) subject global assessment of pain; 4)subject global assessment of disease activity; 5) physician globalassessment of disease activity; 6) subject assessment of physicalfunction (MHAQ); and 7) an acute phase reactant value CRP.

The ACR 20, ACR 50, and ACR 70 definition of response corresponds to a20%, 50%, or 70% improvement, respectively, over baseline in tender andswollen joints (components 1 and 2) and a 20%, 50%, and 70% improvement,respectively, in three of the five remaining core data set measures(components 3 to 7). A Major Clinical Response is defined as maintenanceof an ACR 70 response over a continuous 6-month period. See Table 1 forthe days that data for each component was collected.

The primary efficacy analysis tested for differences in ACR 20 responsebetween the two CTLA4Ig (BMS-188667) treatment groups and the placebogroup at 6 months (Day 180). A sequential testing procedure wasemployed. First, a Chi-square test was used to compare the data for the10 mg/kg CTLA4Ig group with the data for the placebo group at the 0.05level of significance. If this was significant, the data for the 2 mg/kgCTLA4Ig group was compared with the placebo group at the 0.05 level.This testing procedure preserved the overall alpha level at 5%. Similaranalyses were carried out for the ACR 50 and ACR 70 responses at 6months. Differences in ACR 20, ACR 50 and ACR 70 responses between eachCTLA4Ig (BMS-188667) treatment group and the placebo group weresummarized using point estimates and 95% confidence intervals. Subjectswho discontinued the study due to lack of efficacy (i.e., worsening RA)were considered ACR non-responders at all subsequent time points. Forall subjects who discontinued for other reasons, their last ACR responsewas carried forward.

ACR 20, ACR 50, and ACR 70 response rates on Day 360 were comparedbetween each CTLA4Ig (BMS-188667) treatment group and placebo at theDunnett-adjusted 0.027 (two-tailed) level of significance.

The proportion of responders achieving an ACR 20 response at each timepoint was also plotted over time, and the Cochran Mantel-Haenszel test(W. G. Cochran, 1954, Some Methods of Strengthening the CommonChi-Square Test, Biometrics 10:417-451; N. Mantel and W. Haenszel, 1959,Biostatistical Aspects of the Analysis of Data from RetrospectiveStudies of Disease, J Nat Cancer Inst, 22:719-748) was used to comparethe frequency of subjects achieving an ACR 20 response in each CTLA4Ig(BMS-188667) group versus the placebo group.

ACR 20, ACR 50, and ACR 70 responses on Days 15, 30, 60, 90, 120, 150,180, 240, 300, and 360 were also presented for the two CTLA4Ig(BMS-188667) groups and the placebo group. The differences in ACRresponses between the CTLA4Ig (BMS-188667) groups and placebo group weresummarized using 95% confidence intervals. The ACR data plotted overtime were used to assess onset-of-action and to determine time tomaximal response.

A Major Clinical Response was defined as the maintenance of an ACR 70response over a continuous 6-month period. At the 12-month analysis, theproportion of subjects who achieved a Major Clinical Response among thethree groups was summarized.

In order to assess the integrity of the planned analyses, all subjectswho received study medication and discontinued the study for any reasonwere considered ACR non-responders at all scheduled study visitssubsequent to discontinuation.

The cumulative index, ACR-N, was evaluated at each follow-up assessment,and the AUC was assessed for up to 6 months and up to 12 months. Thetrapezoidal rule was used to compute the AUC. The ACR-N AUC was comparedbetween the two CTLA4Ig (BMS-188667) treatment groups and the placebogroup using an analysis of variance (ANOVA) for 6- and 12-month data.This allowed for the assessment of subject response throughout thestudy. These analyses were carried out on the LOCF data sets.

The distributional assumptions regarding the normality of the ACR-N AUCdata were checked using the Shapiro-Wilks test on standardized residualsfrom the ANOVA model at the 10% level of significance.

Surrogate biomarkers were also used to assess the efficacy of theCTLA4Ig+MTX or placebo+MTX treatment regimens. Potential biomarkers forimmunomodulation or inflammation in RA include CRP, soluble IL-2r, RF,soluble ICAM-1, E-selectin, serum IL-6, and TNFα. These parameters weresummarized by treatment group, using frequencies and mean change frombaseline to Day 180 and Day 360.

An Adverse Event (AE) was defined as any new or worsening illness, signsymptom or clinically significant laboratory test abnormality noted bythe Investigator during the course of the study, regardless ofcausality. A serious adverse event (SAE) was defined as an AE that metany of the following criteria: was fatal; was life-threatening; resultedin or prolonged hospitalization; resulted in persistent or significantdisability or incapacity, was cancer, was a congenital anomaly/birthdefect, resulted in an overdose, resulted in the development of drugdependency or drug abuse, or was an important medical event.

Vital sign measurements were obtained at screening and at each studyvisit during and following study drug administration. Vital signmeasurements (seated blood pressure, heart rate, and body temperature)were summarized by treatment group using means.

The two CTLA4Ig (BMS-188667) treatment groups (10 and 2 mg/kg) werecompared with the placebo group. The primary analysis was the comparisonof 6-month ACR response rate for 10 mg/kg and placebo groups, to befollowed by the comparison of 2 mg/kg with placebo only if the formerwas significant. Sample sizes were based on a 5% level (two-tailed) ofsignificance. The ACR 20 response rate for a placebo group at 6 monthswas estimated to be about 25% (Weinblatt M, Kremer J M, Bankhurst A Det. al. A trial of etanercept, a recombinant TNF:Tc fusion protein inpatients with RA receiving methotrexate. NEJM 1999; 340: 253-259). Asample of 107 subjects per treatment group (adjusted for a possible 15%discontinuation rate) was determined to yield a 94% power to detect adifference of 25% at the 5% level (two-tailed). Table 2 summarizes thepower needed to detect the specified treatment differences in ACR 20,ACR 50, and ACR 70 responses at 6 months.

TABLE 2 Response Rates and Power with 107^(a) Subjects per GroupResponse Control Rate (%) Treatment Difference Power (%) ACR 20 25 25 94ACR 50 5 20 95 ACR 70 1 14 90 ^(a)Sample size was adjusted for apossible 15% discontinuation rate; actual sample size was 91.

If the primary comparisons of the 10 mg/kg CTLA4Ig with placebo weresignificant, then for the comparison of the 2 mg/kg CTLA4Ig with placebogroups, the power of the test would be at least 0.88, 0.90, and 0.81 forthe comparison involving ACR 20, ACR 50, and ACR 70 responses at 6months, respectively (Koch D D, Gansky S A. Statistical considerationsfor multiplicity in confirmatory protocols. Drug Info Journal 1996; 30:523-534).

Statistical Analyses

Study Population

Disposition of Subjects

Of 524 subjects who were enrolled in this study, 339 subjects wererandomized: 115 to the 10 mg/kg group; 105 to the 2 mg/kg group; and 119to the placebo group (FIG. 68). The most frequent reason for not beingrandomized was failure to meet inclusion and/or exclusion criteria.

Primary Phase (Days 1-180)

A total of 256 subjects (75.5% of those randomized) completed theprimary phase of the study; 83 subjects discontinued during this period(Table 3). Overall, discontinuation was more than 2-fold higher withplacebo compared with 10 mg/kg CTLA4Ig group. Discontinuation due tolack of efficacy and discontinuation due to an AE were also more than2-fold higher with placebo than with 10 mg/kg CTLA4Ig group.

TABLE 3 Reasons for Discontinuation: Primary Phase (Days 1-180) CTLA4Ig(BMS 188667) 10 mg/kg 2 mg/kg Placebo Total No. Treated, n 115 105 119339 No. Discontinued, n (%) 17 (14.8) 25 (23.8) 41 (34.5) 83 (24.5)Adverse Event 3 (2.6) 7 (6.7) 9 (7.6) 19 (5.6) Lack of Efficacy 12(10.4) 16 (15.2) 28 (23.5) 56 (16.5) Withdrawal of Consent 2 (1.7) 2(1.9) 24 (3.4) 8 (2.4) Completed 180 Days of Therapy, n (%) 98 (85.2) 80(76.2) 78 (65.5) 256 (75.5)Cumulative Discontinuations (Days 1-360)

A total of 235 subjects (69.3% of those randomized) completed bothphases of the study; 104 subjects discontinued by Day 360 (Table 4). Thesame general pattern in disontinuations noted in the primary phase(2-fold higher incidence with placebo compared with 10 mg/kg CTLA4Iggroup) was also observed overall (Days 1-360). This included the overalldiscontinuation rate, discontinuations due to a lack of efficacy anddiscontinuations due to an AE.

TABLE 4 Reasons for Discontinuation: Both Phases (Days 1-360) CTLA4Ig(BMS-188667) 10 mg/kg 2 mg/kg Placebo Total No. Treated, n 115 105 119339 No. Discontinued, n (%) 25 (21.7) 31 (29.5) 48 (40.3) 104 (30.7)Adverse Event 5 (4.3)^(b) 9 (8.6) 11 (9.2) 25 (7.4) Death 0 1 (1.0) 0 1(0.3) Lost to Follow-up 1 (0.9) 2 (1.9) 0 3 (0.9) Other^(a) 1 (0.9) 0 1(0.8) 2 (0.6) Lack of Efficacy 13 (11.3) 17 (16.2) 30 (25.2) 60 (17.7)Withdrawal of Consent 5 (4.3) 2 (1.9) 6 (5.0) 13 (3.8) Completed 360Days of Therapy, n (%) 90 (78.3) 74 (70.5) 71 (59.7) 235 (69.3)^(a)Other reasons for discontinuation were related to compliance^(b)Subject IM101100-32-5 in the 10 mg/kg CTLA4Ig group reported an AEthat was recorded as having resulted in discontinuation from the study;however, this subject was not included in this table.

A Kaplan-Meier plot of the cumulative proportion of subjects whodiscontinued for any reason during the first 12 months is presented inFIG. 69; the cumulative proportion of subjects who discontinued due tolack of efficacy in presented in FIG. 70. Note that in both graphs afterapproximately 30 days of therapy, discontinuation rates with placebowere consistently higher compared with both CTLA4Ig (BMS-188667) groups.Additionally, after approximately 150 days of therapy, discontinuationrates for 2 mg/kg CTLA4Ig group were higher than those for 10 mg/kg.

Demography and Subject Characteristics

Overall, baseline demographic characteristics and baseline clinical RAcharacteristics were generally comparable across the three treatmentgroups and were typical of relatively advanced RA encountered inclinical practice (Table 5 and Table 6). The majority of subjects werewhite females approximately 55 years old with a mean duration of RA ofapproximately 9 to 10 years, a relatively large number of active joints(approximately 29 tender and 21 swollen joints) and visual analoguescores (VAS) approximately 59-65 mm (100 mm scale).

TABLE 5 Baseline Demographic Characteristics CTLA4Ig (BMS-188667) 10mg/kg 2 mg/kg Placebo No. Randomized 115 105 119 Age (yrs) Mean ± SD(Range) 55.8 ± 12.5 54.4 ± 11.3 54.7 ± 12.0 (17, 83) (23, 80) (23, 80)Weight (kg) Mean ± SD (Range) 77.8 ± 18.6 78.7 ± 21.4 79.9 ± 17.6 (40.1, 144.0)  (48.4, 186.8)  (44.0, 140.0) Gender Males, n (%) 29 (25)39 (37) 40 (34) Females, n (%) 86 (75) 66 (63) 79 (66) Race White, n (%)100 (87)  91 (87) 104 (87)  Black, n (%) 6 (5)  0  3 (3) Other, n (%) 9(8) 14 (13) 12 (10) Duration of RA (yrs) Mean ± SD (Range) n = 114^(a) n= 105 n = 117^(a) 9.7 ± 9.8 9.7 ± 8.1 8.9 ± 8.3  (0, 38)  (0, 36)  (0,41)Duration of RA was not reported for 3 subjects.

Although not a component of the ACR criteria, duration of morningstiffness was also assessed and was nearly 2 hours in each of the threegroups. Positive results for RF at baseline were also assessed, and theCTLA4Ig (BMS-188667) treatment groups had higher percentages of subjectswho tested positive for RF (86% for both the 10 mg/kg and 2 mg/kgCTLA4Ig groups compared to 76% for the placebo group).

TABLE 6 Baseline Clinical Rheumatoid Arthritis Characteristics CTLA4Ig(BMS-188667) 10 mg/kg 2 mg/kg Placebo Characteristic (n = 115) (n = 105)(n = 119) Tender Joints, n 115 105 119 Mean ± SD 30.8 ± 12.2 28.2 ± 12.029.2 ± 13.0 Range 11.0, 66.0 3.0, 62.0 4.0, 68.0 Swollen Joints, n 115105 119 Mean ± SD 21.3 ± 8.4 20.2 ± 8.9  21.8 ± 8.8  Range  9.0, 54.04.0, 48.0 8.0, 64.0 Pain (VAS 100 mm), n 113 104 119 Mean ± SD 62.1 ±21.4 64.3 ± 22.3 65.2 ± 22.1 Range  0.0, 99.0  8.0, 100.0 3.0, 95.0Physical Function 115 105 119 (MHAQ 0–3), n Mean ± SD 1.0 ± 0.5 1.0 ±0.5 1.0 ± 0.6 Range 0.0, 2.5 0.0, 2.5  0.0, 2.3  Subject Global 113 105119 Assess (VAS 100 mm), n Mean ± SD 60.1 ± 20.7 59.4 ± 23.7 62.8 ± 21.6Range  10.0, 100.0 8.0, 99.0 4.0, 94.0 MD Global Assess 113 105 119 (VAS100 mm), n Mean ± SD 62.1 ± 14.8 61.0 ± 16.7 63.3 ± 15.5 Range 20.0,98.0 8.0, 95.0 18.0, 93.0  CRP (mg/dL), n 112  99 115 Mean ± SD 2.9 ±2.8 3.2 ± 2.5 3.2 ± 3.2 Range  0.2, 19.9 0.2, 10.8 0.2, 20.9 MorningStiffness 115 103 119 (in minutes), n Mean ± SD 97.9 ± 63.1 104.1 ±63.9  106.0 ± 64.2  Range  0.0, 180.0  0.0, 180.0  0.0, 180.0 RheumatoidFactor  99  90  90 (IU/mL), n % Positive 86% 86% 76%

Baseline demographics and RA characteristics of the overall populationof subjects who had at least one dose of study drug and discontinued dueto lack of efficacy were generally comparable to the entire studypopulation, however, a greater proportion of subjects in thissubpopulation had been diagnosed with RA for >10 years (45%) compared tothe overall study population (34%).

Medical History Findings and Prior Medications

Medical history findings for subjects in this study were consistent withrelatively advanced RA and were generally similar among treatmentgroups. The most frequently occurring findings (in >40% of the subjects)were musculoskeletal findings (not including RA symptoms; 59.3%),gastrointestinal findings (45.1%), and genitourinary findings (42.2%).Other important medical history findings included cardiovascular diseasein approximately 39% of subjects in all treatment groups andendocrine/metabolic findings in approximately 29% of all subjects.

Overall use of MTX, systemic (non-topical) corticosteroids, DMARDs andbiologic RA medications prior to entering the study was generallycomparable across the three treatment groups (Table 7). All subjectswere to have received prior treatment with rheumatic medications,including MTX, to be eligible for the study. Prior treatment with MTXwas not recorded for 4 subjects. Systemic (non-topical) corticosteroiduse prior to randomization was comparable among the three treatmentgroups, with slightly more subjects in the 2 mg/kg CTLA4Ig and placebogroups taking systemic (non-topical) corticosteroids (˜67-68%) comparedto subjects in the 10 mg/kg CTLA4Ig group (60.0%). Use of other DMARDsand biologic RA medications prior to entering the study varied from 0 to12% across treatment groups with no overall predominance in anytreatment group. Mean dosing of MTX and of systemic (non-topical)corticosteroids on Day 1 were comparable among all three treatmentgroups (˜15-16 mg/wk, ˜6-7 mgs/day, respectively.

TABLE 7 Summary of Rheumatic Medications Prior to Enrollment CTLA4Ig(BMS-188667) Prior Rheumatic 10 mg/kg 2 mg/kg Placebo Medication, n(%)^(a) (n = 115) (n = 105) (n = 119) No. Subjects on 114 (99.1) 103(98.1) 118 (99.2) Prior Medications Methotrexate^(b) 114 (99.1) 103(98.1) 118 (99.2) Systemic (non-topical) 69 (60.0) 71 (67.6) 80 (67.2)corticosteroids Other DMARDs 19 (16.5) 19 (18.1) 25 (21.0) Sulfasalazine9 (7.8) 2 (1.9) 10 (8.4) Hydroxychloroquine 8 (7.0) 6 (5.7) 14 (11.8)Cyclosporine 2 (1.7) 4 (3.8) 4 (3.4) Infliximab 2 (1.7) 2 (1.9) 2 (1.7)Etanercept 1 (0.9) 4 (3.8) 1 (0.8) Chloroquine 1 (0.9) 0 0 Leflunomide 02 (1.9) 2 (1.7) ^(a)Categories of prior rheumatic medications were notmutually exclusive. ^(b)Administration of MTX was not recorded for 4subjectsStudy Therapy

Of the treatment groups, the 10 mg/kg CTLA4Ig group had the longest meanduration of exposure for both study phases and the placebo group had theshortest mean duration of exposure for both study phases (Day 180: 163days, 156 days, 140 days; Day 360: 286 days, 268 days, and 234 days; 10mg/kg, 2 mg/kg, and placebo, respectively).

At Day 180 (end of the primary phase), the proportion of subjectsreceiving infusions was higher in the 10 mg/kg CTLA4Ig group (85%)compared with the 2 mg/kg CTLA4Ig group (79%) and the placebo group(66%) (Table 8). At Day 330 (day of last scheduled infusion in thesecondary phase), the proportion of subjects receiving infusions wasalso higher in the 10 mg/kg CTLA4Ig group (78%) compared with the 2mg/kg CTLA4Ig group (70%) and the placebo group (59%).

TABLE 8 Subjects Who Received Infusions on Given Study Days Number (%)of Subjects CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo Day (n = 115)(n = 105) (n = 119) 1 115 (100) 105 (100) 119 (100) 15 114 (99) 104 (99)117 (98) 30 113 (98) 101 (96) 111 (93) 60 108 (94) 97 (92) 103 (87) 90106 (92) 94 (90) 94 (79) 120 100 (87) 86 (82) 83 (70) 150 98 (85) 83(79) 81 (68) 180 98 (85) 83 (79) 78 (66) 210 94 (82) 80 (86) 78 (66) 24095 (83) 78 (74) 76 (64) 270 93 (81) 77 (73) 73 (61) 300 90 (78) 74 (70)72 (61) 330 90 (78) 73 (70) 70 (59)Methotrexate

Subjects were to have been treated with a “stable” dose of MTX (10-30 mgweekly) for at least 6 months, for 28 days prior to Day 1. With theexception of 4 subjects, all subjects received between 10 and 30 mg ofMTX weekly in addition to CTLA4Ig (BMS-188667) during the primary phase(Day 1-180). During the secondary phase (Day 181-360), the dose of MTXcould have been adjusted provided it remained between 10 and 30 mgweekly.

Measurements of Treatment Compliance

During the primary phase, the number of missed infusions of study drugwas ≦2 at any time point (Table 9). During the secondary phase, subjectsin the placebo group appeared to have missed slightly fewer infusionsthan subjects in the CTLA4Ig (BMS-188667) groups. However, more placebothan CTLA4Ig (BMS-188667) subjects discontinued by these later timepoints (see supra).

TABLE 9 Number of Missed Infusions of Study Drug CTLA4Ig (BMS-188667) 10mg/kg 2 mg/kg Placebo (n = 115) (n = 105) (n = 119) Day 1 0 0 0 Day 15 11 0 Day 30 0 1 1 Day 60 0 0 0 Day 90 0 0 0 Day 120 0 1 2 Day 150 1 2 0Day 180 1 0 1 Day 210 4 0 0 Day 240 1 2 1 Day 270 0 2 0 Day 300 1 1 0Day 330 0 0 0Concomitant Therapy

Systemic (non-topical) corticosteroid use was generally comparable amongthe three groups during screening/enrollment (58-67%) and during theprimary phase of the study (67-71%), Tables 10 and 11, respectively.While corticosteroid use decreased in all three treatment groups by Day360, more subjects in the 10 mg/kg CTLA4Ig group took systemic(non-topical) corticosteroids (63.5%) compared to the other twotreatment groups (53.3% and 45.4% for the 2 mg/kg CTLA4Ig and placebogroups, respectively). Several subjects (CTLA4Ig: 0-3%, placebo: 0-10%)received DMARDs other than MTX during screening/enrollment.

TABLE 10 Summary of Rheumatic Concomitant Medications DuringScreening/Enrollment CTLA4Ig (BMS-188667) Rheumatic 10 mg/kg 2 mg/kgPlacebo Medication, n (%)^(a) (n = 115) (n = 105) (n = 119) No. Subjectson 114 (99.1) 103 (98.1) 118 (99.2) Prior Medications Methotrexate 114(99.1) 103 (98.1) 118 (99.2) Systemic (non-topical) 67 (58.3) 70 (66.7)75 (63.0) corticosteroids Other DMARDs 5 (4.3) 6 (5.7) 14 (11.8)Sulfasalazine 3 (2.6) 1 (1.0) 4 (3.4) Hydroxychloroquine 2 (1.7) 3 (2.9)12 (10.1) Cyclosporine 1 (0.9) 1 (1.0) 2 (1.7) Etanercept 0 1 (1.0) 0^(a)Drug categories were not mutually exclusive.

TABLE 11 Subjects Who Received Clinically Relevant ConcomitantMedications During Both Study Phases CTLA4Ig (BMS-188667) 10 mg/kg 2mg/kg Placebo Medication^(a) (n = 115) (n = 105) (n = 119) Systemic(non-topical) 77 (67.0) 71 (67.6) 85 (71.4) corticosteroids (PrimaryPhase) Systemic (non-topical) 73 (63.5) 56 (53.3) 54 (45.4)corticosteroids (Secondary Phase) ^(a)Drug categories were not mutuallyexclusive.Note: Subject IM10100-83-3 (10 mg/kg CTLA4Ig) took mefloquine andsubject IM101100-28-7 (placebo) took quinine between Days 1 and 180;subject IM101100-18-11 (10 mg/kg CTLA4Ig) took quinine between Days 181and 360 as an antimalarial, and was not considered a significantprotocol violation.Efficacy Results

The CTLA4Ig (BMS-188667) 10 mg/kg group had superior efficacy comparedto the placebo group at Day 180 and Day 360. For the 2 mg/kg CTLA4Iggroup, results for some efficacy parameters were significantly bettercompared to the placebo group, results for most other efficacyparameters were numerically higher compared to placebo.

ACR Responses at Day 180

Analysis of the primary efficacy variable for this study, ACR20 responserate at Day 180, showed that the 10 mg/kg CTLA4Ig group wassignificantly (p<0.001) more effective than placebo (Table 12, FIG. 71Aand FIG. 71B).

The ACR50 and ACR70 responses at Day 180 for the 10 mg/kg CTLA4Ig groupwere also significantly higher compared to the placebo group (Table 12,FIG. 71A and FIG. 71B). The ACR50 and the ACR70 responses at Day 180 forthe 2 mg/kg CTLA4Ig group were significantly higher compared to theplacebo group. The ACR20 response at Day 180 for the 2 mg/kg CTLA4Iggroup was slightly higher compared to the placebo group; however, nostatistically significant differences were observed.

TABLE 12 ACR Responses at Day 180 CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kgPlacebo (n = 115) (n = 105) (n = 119) ACR 20 n (%) 70 (60.9) 44 (41.9)42 (35.3) CI 25.6 (12.8, 38.4) 6.6 (−6.2, 19.4) N/A p-value <0.001^(a)0.31 N/A ACR 50 n (%) 42 (36.5) 24 (22.9) 14 (11.8) CI 24.8 (13.8, 35.7)11.1 (1.2, 20.9) N/A p-value <0.001^(a) 0.027^(a) N/A ACR 70 n (%) 19(16.5) 11 (10.5) 2 (1.7) CI 14.8 (7.5, 22.2) 8.8 (2.7, 14.9) N/A p-value<0.001^(a) 0.005^(a) N/A ^(a)Statistically significant difference forthe comparison of BMS-188667 vs placebo.ACR Responses at Day 360

At Day 360, ACR20, ACR50 and ACR70 responses for the 10 mg/kg CTLA4Iggroup were significantly (p<0.001) higher compared to the placebo group(Table 13, FIG. 72A and FIG. 72B). Although the same response rates forthe 2 mg/kg CTLA4Ig group were numerically higher compared to theplacebo group, these differences were not statistically significant.

TABLE 13 ACR Responses at Day 360 CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kgPlacebo (n = 115) (n = 105) (n = 119) ACR 20 N (%) 72 (62.6) 43 (41.0)43 (36.1) CI 26.5 (13.7, 39.3) 4.8 (−7.9, 17.6) N/A P-value <0.001^(a)0.459 N/A ACR 50 N (%) 48 (41.7) 23 (21.9) 24 (20.2) CI 21.6 (9.7, 33.4)1.7 (−8.9, 12.4) N/A P-value <0.001^(a) 0.75 N/A ACR 70 N (%) 24 (20.9)13 (12.4) 9 (7.6) CI 13.3 (4.4, 22.2) 4.8 (−3.0, 12.6) N/A P-value0.003^(a) 0.227 N/A ^(a)Statistically significant difference for thecomparison of 10/mg/kg CTLA4Ig group vs placebo.ACR Responses by Visit

For the comparison of the 10 mg/kg CTLA4Ig group to the placebo group,statistically significant improvements were observed for all threeresponse rates (ACR 20, ACR 50, and ACR 70) by Day 90, and these valuesremained statistically significant at every time point up to andincluding Day 360 (p≦0.008 for all three ACR response rates) (FIG. 73A,FIG. 73B, and FIG. 73C). In fact, statistically significant improvementsin ACR 50 and ACR 70 response for the 10 mg/kg CTLA4Ig group occurred asearly as Day 30 (p=0.039 and p=0.04, respectively).

For the 2 mg/kg CTLA4Ig group, statistically significant improvementscompared to placebo were observed in ACR 50 and ACR 70 responses at Day180 (p=0.027 and p=0.005, respectively). At Day 360, improvements in ACRresponse were slightly greater in the 2 mg/kg CTLA4Ig group compared tothe placebo group; however, no statistically significant differenceswere observed.

After adjusting for visit using the Cochran-Mantel Haenszel test, asignificant difference in ACR 20 response was observed for the 10 mg/kgCTLA4Ig group compared to the placebo group at both Day 180 and Day 360.No significant difference was observed between the 2 mg/kg CTLA4Ig andplacebo groups at both timepoints. Similar results were obtained for ACR50 response at both time points. For ACR 70 response at both timepoints, a significant difference was observed for both CTLA4Ig(BMS-188667) treatment groups compared to the placebo group.

Summary of Major Clinical Response

Major Clinical Response was defined as maintenance of an ACR 70 responseover a continuous 6-month period. The percentages of subjects whoachieved a Major Clinical Response at Day 360 were significantly higherin both the 10 mg/kg and 2 mg/kg CTLA4Ig groups (7.8% and 5.7%,respectively) when compared to the placebo group (0.8%; p=0.008 and0.036, respectively) (Table 14).

TABLE 14 Summary of Major Clinical Response by Day 360 CTLA4Ig(BMS-188667) 10 mg/kg 2 mg/kg Placebo (n = 115) (n = 105) (n = 119) No.Subjects with 9 (7.8) 6 (5.7) 1 (0.8) a Major Response Diff (CI) 7.0(1.8, 12.2) 4.9 (0.3, 9.4) N/A p-value 0.008^(a) 0.036^(a) N/A^(a)Indicates a statisticall significant difference for the comparisonof BMS-188667 vs placebo.Mean Numeric ACR (ACR-N) and ACR-N Area Under the Curve (ACR-N-AUC)

Overall, mean numeric ACR (ACR-N) for all treatment groups increasedover time during the first 6 months of the study (FIG. 74). During thesecond 6 months, mean ACR-N increased slightly with 10 mg/kg CTLA4Ig,but remained relatively unchanged with 2 mg/kg CTLA4Ig and placebo. Ateach study visit, the ACR-N was consistently higher for the 10 mg/kgCTLA4Ig group compared to the 2 mg/kg CTLA4Ig and placebo groups.

Compared to the placebo group, the differences in values for ACR-N AUC(area under the curve) for the 10 mg/kg CTLA4Ig group was significantly(p<0.001) higher by Day 360.

Percentage Improvement from Baseline at Day 180

For the 10 mg/kg CTLA4Ig group, improvements in each individual ACRcomponent (tender and swollen joint counts, CRP, pain, subject globalassessment, physician global assessment, and physical function) at Day180 were statistically significant relative to improvements for theplacebo group (Table 15).

For the 2 mg/kg CTLA4Ig group, statistically significant improvementscompared to the placebo group were observed in physician globalassessment and CRP at Day 180. Furthermore, CRP levels in the placebogroup actually worsened at Day 180. Change from baseline in meanduration of morning stiffness was comparable among the three treatmentgroups at Day 180.

TABLE 15 Mean Percentage Improvement from Baseline at Day 180(Individual Components of ACR Criteria) CTLA4Ig (BMS-188667) 10 mg/kg 2mg/kg Placebo Component (n = 115) (n = 105) (n = 119) Tender Joints n =114 n = 104 n = 118 Mean % Change 59.78* 43.15  31.88 Swollen Joints n =114 n = 104 n = 118 Mean % Change 55.28* 45.34*  33.49 CRP n = 108 n =98 n = 114 Mean % Change 31.79* 16.41* −23.43 Pain n = 109 n = 102 n =118 Mean % Change 46.19* 22.09*  8.20 Subject Global n = 111 n = 103 n =118 Assessment Mean % Change 40.76*  9.07  17.48 MD Global n = 111 n =103 n = 116 Assessment Mean % Change 51.91* 38.71*  25.14 PhysicalFunction n = 107 n = 98 n = 110 Mean % Change 41.21* 21.63  13.71Duration Morning n = 98 n = 82 n = 80 Stiffness Mean ± SD (minutes) 61.9± 55.4 60.8 ± 66.1 55.9 ± 66.2 *Indicates p < 0.05 in comparison withplacebo since 95% CIs did not include zeroPercentage Improvement from Baseline at Day 360

For the 10 mg/kg CTLA4Ig group, improvements in each individual ACRcomponent (tender and swollen joint counts, CRP, pain, subject globalassessment, physician global assessment, and physical function) at Day360 were statistically significant relative to improvements for theplacebo group. Mean percentage improvements from baseline to Day 360 arepresented in Table 16 for all clinical parameters of the ACR criteria.

For the 2 mg/kg CTLA4Ig group, statistically significant improvementscompared to the placebo group were observed in physician globalassessment and CRP at Day 360. Furthermore, CRP levels in the placebogroup actually worsened at Day 360. At Day 360, the CTLA4Ig (BMS-188667)treatment groups had greater changes from baseline in duration ofmorning stiffness compared to the placebo group.

TABLE 16 Mean Percentage Improvement from Baseline at Day 360(Individual Components of ACR Criteria) CTLA4Ig(BMS-188667) 10 mg/kg 2mg/kg Placebo Component (n = 115) (n = 105) (n = 119) Tender Joints n =115 n = 105 n = 119 Mean % Change 66.39* 43.54*  29.97 Swollen Joints n= 115 n = 105 n = 119 Mean % Change 59.74* 46.40  36.17 CRP n = 112 n =98 n = 115 Mean % Change 27.59* 10.31* −31.26 Pain n = 112 n = 104 n =119 Mean % Change 44.93* 26.26  12.55 Subject Global n = 113 n = 105 n =119 Assessment Mean % Change 41.01* 16.08  1.99 MD Global n = 113 n =105 n = 119 Assessment Mean % Change 53.48* 37.87*  24.14 PhysicalFunction n = 109 n = 100 n = 111 Mean % Change 42.32* 22.94  10.25Duration Morning n = 88 n = 71 n = 72 Stiffness Mean ± SD 66.2 ± 59.5*66.6 ± 72.2 49.7 ± 73.9 *Indicates p < 0.05 in comparison with placebosince 95% CIs did not include zeroNew Active Joints

The proportion of new active joints was determined using the validated28-joint count (out of 68 total tender joints and out of 66 totalswollen joints) proposed by Smollen et al (Smollen J S, Breedveld F C,Eberl G, Jones I et al. Validity and reliability of thetwenty-eight-joint count for the assessment of RA activity. Arthritis &Rheum 1993; 38: 38-43). The proportion of new active joints (both tenderand swollen) at Day 180 was lowest for subjects receiving 10 mg/kgCTLA4Ig (FIG. 75).

At Day 180, the percentages of subjects reporting no new tender jointsand no new swollen joints was highest in the 10 mg/kg CTLA4Ig group(FIG. 76A, FIG. 77A). The percentage of subjects who reported no newtender joints and no new swollen joints was approximately 59% and 52%,respectively, in the 10 mg/kg CTLA4Ig group; 38% and 44%, respectively,in the 2 mg/kg CTLA4Ig group; and 41% and 37%, respectively, in theplacebo group.

The proportion of new active joints (both tender and swollen) at Day 360was lowest for subjects receiving 10 mg/kg CTLA4Ig (FIG. 78). Thispattern for the proportion of new active joints mirrored the patternseen at Day 180.

Similarly, at Day 360, the proportion of subjects reporting no newtender joints and no new swollen joints was highest in the 10 mg/kgCTLA4Ig group (FIG. 76B, and FIG. 77B). The percentage of subjects whoreported no new tender and no new swollen joints was approximately 71%and 61%, respectively, in the 10 mg/kg CTLA4Ig group; 41% and 44%,respectively, in the 2 mg/kg CTLA4Ig group; and 42% for both counts inthe placebo group.

Improvement in Clinical Parameters Among Subjects with an ACR Response

Among ACR 20, ACR 50, and ACR 70 responders, improvement in the corecomponents of the ACR criteria were slightly greater for the two CTLA4Ig(BMS-188667) treatment groups compared to placebo.

The onset of action for subjects who received the 10 mg/kg CTLA4Ig doseoccurred after approximately 15 days, with significant increases in ACR20 improvement occurring at ≧Day 60 for ACR50 at ≧Day 90 for ACR70 at≧Day 30 and in each instance, continuing until Day 360 (see FIG. 73A,FIG. 73B and FIG. 73C).

Changes from Baseline for the Health Outcomes Short Form Questionnaire(SF-36)

The impact of CTLA4Ig (BMS-188667) on health-related quality of life wasassessed using the Health Outcomes Short Form Questionnaire SF-36(summary scores range from 0 to 100 with higher scores indicating abetter quality of life). Analyses were performed on the LOCF (lastobservation carried forward) data set as well as the as the observeddata set.

For the 10 mg/kg CTLA4Ig group, statistically significant improvementfrom baseline compared to the placebo group was observed in all fourmental health and all four physical health domains of the SF-36 at Day180, using the LOCF analysis (i.e., 95% CIs did not include 0) (FIGS.79A, 79B). For the 2 mg/kg CTLA4Ig group, there were numericalimprovements in the mental health or physical health domains compared toplacebo at Day 180, however, these improvements were not statisticallysignificant.

Results of analyses performed on the as-observed data set were similarto those observed for the LOCF data set except that the “role emotional”domain at Day 180 was not significantly improved (but was numericallyimproved) for the comparison between the 10 mg/kg CTLA4Ig and placebogroups using the as-observed data set.

The physical component and the mental health component summary measuresat Day 180 are shown in Table 17.

TABLE 17 Mean Change from Baseline to Day 180 for the SF-36 (Physicaland Mental Health Components) CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kgPlacebo Summary Score (n = 115) (n = 105) (n = 119) Mental HealthComponent n = 115 n = 103 n = 118 Baseline Mean 44.52 43.06 41.75Postbaseline Mean 48.69 45.59 44.04 Mean Change from 4.17 2.53 2.30Baseline 95% CI (2.46, 5.88) (0.39, 4.67) (0.42, 4.17) PhysicalComponent n = 115 n = 103 n = 118 Baseline Mean 31.13 30.80 32.33Postbaseline Mean 39.30 35.47 35.21 Mean Change from 8.16 4.67 2.88Baseline 95% CI (6.33, 9.99) (3.25, 6.09) (1.54, 4.22)

Results of the Health Outcomes at Day 360 were similar to those seen atDay 180. For the 10 mg/kg CTLA4Ig group, statistically significantimprovements from baseline compared to the placebo group were observedin all four mental and all four physical domains of the SF-36 at Day360, using the LOCF analysis (i.e., 95% CIs did not include 0) (FIGS.80A, and 80B). For the 2 mg/kg CTLA4Ig group, a statisticallysignificant difference in three of four physical domains at Day 360 andone of four mental domains at Day 360 compared to the placebo group wasobserved.

Results of analyses performed on the as-observed data set were similarto those observed for the LOCF data set.

The physical component and mental health component summary measures atDay 360 is shown in Table 18.

TABLE 18 Mean Change from Baseline to Day 360 for the SF-36 (Summariesof Physical Component and Mental Health Component) CTLA4Ig (BMS-188667)10 mg/kg 2 mg/kg Placebo Summary Score (n = 115) (n = 105) (n = 119)Mental Health Component n = 115 n = 103 n = 118 Baseline Mean 44.5243.06 44.75 Postbaseline Mean 48.83 45.65 43.22 Mean Change from 4.312.59 1.47 Baseline 95% CI (2.64, 5.98) (0.64, 4.55) (−0.14, 3.08) Physical Component n = 115 n = 103 n = 118 Baseline Mean 31.13 30.8032.33 Postbaseline Mean 38.93 36.49 34.93 Mean Change from 7.79 5.692.60 Baseline 95% CI (5.90, 9.68) (4.10, 7.28) (1.09, 4.11)Biomarker and Pharmacodynamic Data

There were significant improvements (decreases) in 5 of the 6biomarker/pharmacodynamic (PD) parameters with 10 mg/kg CTLA4Ig at Day180 (soluble IL-2r, rheumatoid factor (RF), ICAM-1, E-selectin and IL-6)and a numerical decrease in TNF-α (Table 19). There were significantimprovements (decreases) in 3 of the 6 biomarker/PD parameters with 2mg/kg CTLA4Ig at Day 180 (soluble IL-2r, RF and IL-6) and a numericalimprovement in ICAM-1. There were no significant changes in any of thebiomarker/PD parameters with placebo at Day 180. There appears to be adose response relationship with the improvements (decreases) inbiomarker/PD parameters.

TABLE 19 Pharmacodynamic Measures at Day 180 CTLA4Ig (BMS-188667) 10mg/kg 2 mg/kg Placebo Parameter (n = 115) (n = 105) (n = 119) SolubleIL-2r n = 95 n = 84 n = 76 (Normal range: 640–2543 pg/mL) Baseline Mean(±SD) 1426.19 ± 751.76  1396.82 ± 610.21  1429.13 ± 667.84  PostbaselineMean (±SD) 1112.62 ± 699.68  1261.31 ± 473.66  1470.03 ± 637.75  MeanChange −316.23 −135.51 43.59 95% CI (−417.73, −214.72) (−241.48,−29.53)  (−71.24, 158.43) Rheumatoid Factor n = 95 n = 84 n = 74 (NormalRange: 0–20 IU/mL) Baseline Mean (±SD) 289.71 ± 401.95 256.19 ± 307.92196.11 ± 265.48 Postbaseline Mean (±SD) 185.43 ± 269.52 227.82 ± 276.27204.36 ± 320.09 Mean Change −104.27 −28.12 −0.62 95% CI (−151.53,−57.01)  (−52.13, −4.11)  (−31.67, 30.43)  ICAM-1 n = 95 n = 82 n = 75Baseline Mean (±SD) 404.89 ± 137.72 393.47 ± 150.85 387.33 ± 230.93Postbaseline Mean (±SD) 364.74 ± 109.47 387.25 ± 142.73 386.17 ± 163.82Mean Change −40.42 −6.22 1.09 95% CI (−58.06, −22.78) (−27.49, 15.05) (−31.88, 34.05)  E-selectin n = 89 n = 80 n = 71 Baseline Mean (±SD)68.07 ± 32.93 67.32 ± 37.13 68.23 ± 43.09 Postbaseline Mean (±SD) 61.01± 31.53 67.86 ± 40.20 67.37 ± 35.66 Mean Change −8.41 0.54 −0.68 95% CI(−13.24, −3.58)  (−5.95, 7.03)  (−6.87, 5.51)  Serum IL-6 n = 86 n = 74n = 69 (Normal Range: 0.3–14.8 pg/mL) Baseline Mean (±SD) 28.47 ± 38.2831.75 ± 42.29 21.20 ± 26.51 Postbaseline Mean (±SD)  9.25 ± 15.85 16.00± 22.13 23.98 ± 37.92 Mean Change −20.30 −16.10 1.98 95% CI (−27.55,−13.06) (−24.20, −8.00)  (−7.21, 11.17) TNFα n = 84 n = 74 n = 69(1.2–8.0 pg/mL) Baseline Mean (±SD) 11.17 ± 23.72  7.51 ± 13.25 13.12 ±23.20 Postbaseline Mean (±SD) 7.57 ± 7.90 6.20 ± 4.48  9.59 ± 11.21 MeanChange −3.66 −1.21 −3.54 95% CI (−8.62, 1.30)  (−4.32, 1.90)  (−7.82,0.75) 

Overall, the pattern in the changes in biomarker/PD data at Day 360 weresimilar to that seen at Day 180. There were significant improvements(decreases) in 5 of the 6 biomarker/PD parameters with 10 mg/kg CTLA4Igat Day 360 (soluble IL-2r, RF, ICAM-1, E-selectin and IL-6) and anumerical, but not statistically significant improvement observed forTNF-α (Table 20). There was a significant improvement (decrease) in IL-6only with 2 mg/kg CTLA4Ig at Day 360, however, numerical improvementswere seen with RF and ICAM-1. There were no significant changes in anyof the biomarker/PD parameters with placebo at Day 360. As seen with Day180 data, it appeared that all of the improvements (decreases) inbiomarker/PD parameters occurred in a dose response manner.

A comparison of the postbaseline means for the biomarker/PD parametersat Day 180 to those at Day 360 reveals important trends. For the 10mg/kg CTLA4Ig group, all biomarkers/PD measures continued to decrease,with the exception of soluble IL-2r which increased slightly. For the 2mg/kg CTLA4Ig group, mean values for 3 of the biomarkers/PD parameterseither decreased slightly (ICAM-1, serum IL-6) or remained relativelyconstant (E-selectin) and mean values for the other 3 biomarkers/PDmeasures increased slightly (soluble IL-2r, RF, TNF α). For the placebogroup, mean values for all of the biomarkers/PD parameters increasedslightly at Day 360, with the exception of TNF a which remainedrelatively unchanged.

TABLE 20 Pharmacodynamic Measures at Day 360 CTLA4Ig (BMS-188667) 10mg/kg 2 mg/kg Placebo Measure (n = 115) (n = 105) (n = 119) SolubleIL-2r n = 68 n = 56 n = 55 (Normal range: 640–2543 pg/mL) Baseline Mean(±SD) 1372.10 ± 770.11  1373.86 ± 567.75  1459.93 ± 695.07  PostbaselineMean (±SD) 1185.51 ± 638.95  1413.84 ± 452.50  1666.59 ± 611.97  MeanChange −194.31 39.99 206.22 95% CI (−305.67, −82.96)  (−69.87, 149.84) (35.88, 376.56) Rheumatoid Factor n = 69 n = 55 n = 58 (Normal Range:0–20 IU/mL) Baseline Mean (±SD) 261.43 ± 333.58 258.42 ± 318.65 179.12 ±207.72 Postbaseline Mean (±SD) 143.13 ± 180.80 236.61 ± 287.36 206.42 ±256.27 Mean Change −118.30 −25.64 20.90 95% CI (−175.19, −61.42) (−58.50, 7.23)  (−10.72, 52.51)  ICAM-1 n = 77 n = 68 n = 64 BaselineMean (±SD) 406.44 ± 145.22 393.41 ± 132.97 405.67 ± 245.16 PostbaselineMean (±SD) 354.90 ± 111.40 380.42 ± 113.20 405.07 ± 194.15 Mean Change−55.15 −12.98 1.47 95% CI (−74.80, −35.49) (−35.36, 9.39)  (−26.41,29.35)  E-selectin n = 75 n = 68 n = 62 Baseline Mean (±SD) 68.84 ±34.38 66.75 ± 37.10 69.72 ± 44.38 Postbaseline Mean (±SD) 58.77 ± 26.6167.58 ± 31.50 71.90 ± 47.43 Mean Change −10.89 0.83 2.34 95% CI (−15.70,−6.08)  (−5.62, 7.28)  (−4.53, 9.20)  Serum IL-6 n = 56 n = 47 n = 48(Normal Range: 0.3–14.8 pg/mL) Baseline Mean (±SD) 27.68 ± 38.56 27.19 ±32.45 17.27 ± 22.47 Postbaseline Mean (±SD)  7.64 ± 14.21 13.93 ± 19.0017.72 ± 29.76 Mean Change −20.88 −12.72 −0.19 95% CI (−31.56, −10.19)(−22.49, −2.94)  (−7.55, 7.18)  TNFα n = 61 n = 48 n = 50 (1.2–8.0pg/mL) Baseline Mean (±SD)  9.71 ± 22.80 6.27 ± 3.62 10.81 ± 21.24Postbaseline Mean (±SD) 6.67 ± 4.80 7.18 ± 8.14  9.36 ± 26.43 MeanChange −3.02 1.08 −1.41 95% CI (−8.70, 2.67)  (−1.26, 3.42)  (−5.14,2.33) 

The data are shown graphically for these biomarker/PD measures, as wellas for changes in CRP levels, in FIGS. 81 through 87.

In order to assess the integrity of the planned analyses, all subjectswho received study medication and discontinued the study for any reasonwere considered ACR non-responders at all scheduled study visitssubsequent to discontinuation. Results of these analyses (Table 21) wereconsistent with the efficacy results already presented. The proportionof subjects who received 10 mg/kg CTLA4Ig and achieved an ACR 20, ACR50, or ACR 70 response at Day 180 was significantly (p<0.001) highercompared to the proportion of subjects who received placebo. For the 2mg/kg CTLA4Ig group, a significantly (p≦0.009) higher proportion ofsubjects achieved either an ACR 50 or ACR 70 response.

TABLE 21 ACR Response at Day 180 (Non-Completer Equals Non-Responder)CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo (n = 115) (n = 105) (n =119) ACR 20, n (%) 67 (58.3) 41 (39.0) 38 (31.9) Diff (CI) 26.3 (13.6,39.1) 7.1 (−5.4, 19.7) N/A p-value <0.001^(a) 0.266 N/A ACR 50, n (%) 41(35.7) 24 (22.9) 12 (10.1) Diff (CI) 25.6 (14.8, 36.3) 12.8 (3.1, 22.4)N/A p-value <0.001^(a) 0.009^(a) N/A ACR 70, n (%) 19 (16.5) 11 (10.5) 2(1.7) Diff (CI) 14.8 (7.5, 22.2) 8.8 (2.7, 14.9) N/A p-value <0.001^(a)0.005^(a) N/A ^(a)Indicates a statistically significant difference forthe comparison of BMS-188667 vs placebo.

In addition, all primary efficacy analyses were performed on the WOCF(worst observation carried forward) data set. ACR responses based on theWOCF data set were slightly lower than those reported in Table 13 andwere comparable to those presented in Table 21. These findings confirmthe consistency of ACR response rates in the CTLA4Ig (BMS-188667)treatment groups.

The dosages of anti-rheumatic concomitant medications were to becollected to assess the need for these medications at 6 and 12 months;however, the available data were inadequate to perform these analyses.Only baseline values for mean dose of methotrexate and systemic(non-topical) corticosteroids are provided.

Efficacy Conclusions

CTLA4Ig (BMS-188667) administered at 10 mg/kg (+MTX) had superiorefficacy compared to placebo (+MTX) at Day 180 and Day 360. For thefollowing efficacy parameters, administration of 10 mg/kg CTLA4Ig wassignificantly better than placebo:

-   -   Primary efficacy variable: ACR20 response at Day 180 (p<0.001)    -   ACR50 and ACR70 responses at Day 180 (p<0.001)    -   ACR20, ACR50 and ACR70 responses at Day 360 (p≦0.003)    -   Statistically significant differences in ACR50 and ACR70        responses observed by Day 30 (p=0.039 and p=0.04), statistically        significant differences in all 3 response rates (ACR 20, ACR50        and ACR70) observed by Day 90; these values remained        statistically significant at every timepoint up to and including        Day 360 (p≦0.008)    -   Proportions of subjects who achieved a Major Clinical Response        (maintenance of an ACR 70 response over a continuous 6-month        period) at Day 360 (p=0.008)    -   Mean numeric ACR-AUC by Day 360 (p<0.001)    -   Mean percentage improvements in each individual ACR component at        Day 180 and Day 360 (p<0.05, 95% CIs did not include 0)    -   Improvements in all four mental and all four physical domains of        the Health Outcomes evaluation (SF-36) at both Day 180 and Day        360 (p<0.05, 95% CIs did not include 0)

In addition to the above statistically significant differences, the 10mg/kg CTLA4Ig group had a lower number of new active joints and a highernumber of subjects reporting no new active tender and swollen jointscompared with the placebo group at Day 180 and at Day 360.

Significant improvement with 10 mg/kg CTLA4Ig compared with placebo wasseen in nearly all measured pharmacodynamic paramenters (soluble IL12r,RF, ICAM-1, E-selectin and IL-6) and numerical improvement in TNF-α upto 1 year.

For the 2 mg/kg CTLA4Ig group, some efficacy parameters weresignificantly better compared to the placebo group:

-   -   ACR50 response at Day 180 (p=0.027)    -   ACR70 response at Day 180 (p=0.005)    -   Statistically significant differences in ACR70 observed by Day        60 (p=0.032) and statistically significant differences in ACR 50        and ACR 70 at Day 180 (p=0.027 and p=0.005)    -   Proportions of subjects who achieved a Major Clinical Response        (maintenance of an ACR 70 response over a continuous 6-month        period) at Day 360 (p=0.036)    -   Mean percentage improvements in some of the individual ACR        component at Day 180 and Day 360 (p<0.05, 95% CIs did not        include 0)

For many other efficacy parameters, 2 mg/kg CTLA4Ig was numericallybetter than placebo.

Safety Results

Overall, the safety profile of CTLA4Ig (BMS-188667) was similar toplacebo. There were no major safety problems.

Clinical Laboratory Evaluation

Overall, no new safety issues emerged from the evaluation of meanchanges in laboratory values. Mean values for hemoglobin, WBCs,neutrophils, platelets, ALT, AST, GGT and total protein were within thenormal range at baseline and remained within the normal range during thestudy. In general, results of the laboratory tests did not revealconsistent out-of range values or abnormal trends that could beattributed to study medication.

Vital Signs, Physical Findings, and Observations Related to Safety

On each day of study drug administration, vital signs (body temperature,heart rate, and seated blood pressure) were monitored pre-dose and at15, 30, 45, 60, 75, 90 and 120 minutes post-infusion. Overall, meanvalues for all vital sign parameters were within normal range and stablethroughout the 360-day study period for all treatment groups.

Example 8

While established animal models that mimic acute coronary syndrome inhumans do not exist, the use of CTLA4Ig or L104EA29YIg to inhibitatherosclerosis in animal models may be possible. Using murine CTLA4Ig,the ability to suppress atherosclerosis in apolipoprotein E null (apoE−/−) mice maintained on a high fat diet and with and without a stimulusto promote inflammation (i.e., infectious agents, cytokineadministration) is explored. It is anticipated that CTLA4Ig may affordreduction of atherosclerotic plaque formation in this animal model ascompared with mice receiving placebo (sham) injections.

Example 9

The potential use of CTLA4Ig or L104EA29YIg is further explored inseveral patient populations.

Patients presenting to the hospital with unstable angina ornon-ST-segment elevation myocardial infarction (non-STEMI) and evidenceof underlying inflammation (i.e., elevated hsCRP) are randomized toreceive either placebo or CTLA4Ig/L104EA29YIg. Patients receive abackground of standard medical or interventional therapies as ethicallymandated and consistent with the most recent American HeartAssociation/American College of Cardiology Guidelines. The primaryendpoint would be a clinical composite reflecting a reduction insubsequent patient morbidity and mortality and/or a redcution ofsubsequent risk reduction as measred by hsCRP (or other infalmmatorymarker[s]).

1. A method for treating a cardiovascular disease comprisingadministering to a subject in need thereof an effective amount of asoluble CTLA4 molecule comprising an extracellular domain of a CTLA4molecule, or portion thereof which binds a B7-1 or B7-2 antigenexpressed on activated B cells, wherein the extracellular domain of theCTLA4 molecule comprises the amino acids shown in SEQ ID NO: 17beginning with methionine at position +1 or with alanine at position −1and ending with aspartic acid at position 124, wherein thecardiovascular disease is atherosclerosis, and wherein the method doesnot comprise administering a TNF/TNFR blocking molecule, soluble gp39,soluble CD40, antibodies which bind to CD40, or monoclonal antibodies togp39.
 2. The method of claim 1, wherein said cardiovascular disease isassociated with at least one marker of inflammation selected from thegroup consisting of CRP, hsCRP, IL-10, CD40L, sCD40L, IL-6, sICAM-1,TNF-α, white blood cell count, fibrinogen, and serum amyloid A.
 3. Themethod of claim 2, wherein said marker of inflammation is selected fromthe group consisting of CRP, hsCRP, IL-6, and TNF-α.
 4. The method ofclaim 1, wherein the effective amount the soluble CTLA4 molecule isabout 0.1 to 100 mg/kg weight of the subject, about 0.5 to 100 mg/kgweight of a subject 0.5 to 5 mg/kg weight of a subject, about 5 to 10mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject,about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight ofa subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject,about 40 to 45 mg/kg of a subject, about 45 to 50 mg/kg weight of asubject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kgweight of a subject, about 60 to 65 mg/kg weight of a subject, about 65to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of asubject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kgweight of a subject, about 85 to 90 mg/kg weight of a subject, about 90to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of asubject, about 2 to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kgweight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of asubject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject,about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kgweight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of asubject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject,about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kgweight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of asubject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of asubject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kgweight of a subject, about 12 to 14 mg/kg weight of a subject, about 14to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of asubject, about 18 to 20 mg/kg weight of a subject, about 0.5 mg/kgweight of the subject, 2 mg/kg weight of the subject, 10 mg/kg weight ofthe subject, about 0.5 mg/kg to 100 weight of the subject, about 0.5 to10 mg/kg weight of a subject, about 0.1 to 20 mg/kg weight of a subject,about 500 mg for a subject weighing less than 60 kg, 750 mg for asubject weighing between 60-100 kg or 1000 mg for a subject weighingmore than 100 kg.
 5. The method of claim 1, wherein the soluble CTLA4molecule is a CTLA4 fusion molecule.
 6. The method of claim 5, whereinthe CTLA4 fusion molecule comprises the extracellular domain of theCTLA4 molecule, or portion thereof which binds a B7-1 or B7-2 antigenexpressed on activated B cells, joined to a non-CTLA4 molecule.
 7. Themethod of claim 6, wherein the non-CTLA4 molecule comprises an aminoacid sequence which alters the solubility or affinity of the solubleCTLA4 molecule.
 8. The method of claim 7, wherein the amino acidsequence which alters the solubility or affinity comprises animmunoglobulin moiety.
 9. The method of claim 8, wherein theimmunoglobulin moiety comprises one or more mutations to alter effectorfunction.
 10. The method of claim 8, wherein the immunoglobulin moietycomprises a hinge and any or all of the cysteine residues within thehinge are substituted with serine.
 11. The method of claim 8, whereinthe immunoglobulin moiety is an immunoglobulin constant region orportion thereof.
 12. The method of claim 11, wherein the immunoglobulinconstant region or portion thereof is mutated to alter effectorfunction.
 13. The method of claim 11, wherein the immunoglobulinconstant region comprises a hinge, CR2 and CR3 regions of animmunoglobulin molecule.
 14. The method of claim 11, wherein theimmunoglobulin constant region or portion thereof is a human or monkeyimmunoglobulin constant region or portion thereof.
 15. The method ofclaim 12, wherein the immunoglobulin constant region comprises a hinge,CH2 and CH3 regions of an immunoglobulin molecule.
 16. The method ofclaim 8, wherein the CTLA4Ig is shown in SEQ ID NO: 19 beginning withmethionine at position +1 or with alanine at position −1 and ending withlysine at position +357.
 17. The method of claim 16, wherein saidcardiovascular disease is associated with at least one marker ofinflammation selected from the group consisting of CRP, hsCRP, IL-6, andTNF-α.
 18. The method of claim 1, wherein the soluble CTLA4 molecule isadministered in combination with at least one additional therapeuticagent that is not a TNF/TNFR blocking molecule.